The adaptation of the frog tongue to bitter solutions: Enhancing effect on gustatory neural response...

THE ADAPTATION OF THE FROG TONGUE TO BITTER SOLUTIONS: ENHANCING EFFECT ON GUSTATORY

NEURAL RESPONSE TO SALT STIMULI

TOSHIHIDE SATO and KUMII~O SUGIMOTO

Department of Physiology, School of Dentistry, Tokyo Medical and Dental University. Bunkyo-ku. Tokyo 113. Japan

(Rrcritsed 12 June 1978)

Abstract-l. The initial phasic component of frog gustatory neural responses to various 0.1 M salt solutions was greatly augmented in amplitude after the tongue was adapted for IO set to 1 mM quinine- HCI (Q-HCI). quinine-H,SO, (Q-H2SOI) and picric acid.

2. Out of 103 examined gustatory units responding to both 0.5 M NaCl and I mM Q-HCI or to 0.5 M NaCI alone. 67% exhibited an enhancement of response to the NaCl after the Q-HCI adaptation but the remaining 33% showed a suppression or no alteration of NaCl response after the Q-HCI.

3. Intracellular taste cell responses to salt stimuli after I mM Q-HCI adaptation showed an initial phasic depolarization which was not observed under control Ringer adaptation. This depolarization might be concerned with the enhancement of initial phasic neural responses to salts following the Q-HCI adaptation

INTRODUCTION

It has been demonstrated that gustatory neural responses to taste stimuli are potentiated or depressed when the tongue is adapted to certain taste solutions prior to a test (Yinon & Erickson, 1970; Smith & Frank. 1972: Akaike & Sato, 1976). In the preceding papers. Sato (1976b. 1978) showed that a frog gustatory neural response to NaCl is greatly enhanced by adapting the tongue to bitter-tasting substances including Q-HCI for a short time. Therefore, it is very much likely that frog gustatory neural responses to other salt stimuli may be potentiated after adaptation to various bitter solutions.

The purpose of the present experiments is to inves- tigate the properties and mechanisms of the salt response enhancement following bitter adaptation by recording electrical activities from gustatory nerves. units and cells. Brief reports of a preliminary series of experiments. not included in this paper, have appeared elsewhere (Sato. 1974. 1975; Sato & Sugi- moto. 1977).

MATERIALS AND METHODS

Twenty-three adult bullfrogs (Ram carrshaianu) were used. The animal was anesthetized with an i.p. injection of a 50”” (w/v) urethane-Ringer solution (2 g/kg body wt). The glossopharyngeal nerve of either side was exposed free from the surrounding connective tissues and cut near the hyoid bone. To avoid the muscular contraction of the tongue the hypoglossal nerves and the hyoglossal muscles of both sides were cut. Almost the entire tonque was pinned to a cork plate placed on an experimental chambe;.

The electrical activities of whole glossopharyngeal nerves, gustatory units, and gustatory cells were reco;ded in situ with silver wire electrodes, suction electrodes and glass capillary microelectrodes, respectively. The methods and procedures for recordings were the same as those described previously (Sato. 1978). The taste solutions were delivered to the tongue surface using a semiautomatically controlled gustatory stimulator (Sato, 1972). The taste stimuli cmploqed were as follows: salt stimuli: NaCI. LiCI.

KCI. NH,CI. KNO,. Na:SOa. M&II. K?SO,. CaC12. NaHC03. and NaHZPO,: bitter stimuli: Q--HCI. Q--H2S04. picric acid. brucine. caffeine and nicotine. Unless otherwise stated. all the chemicals were dissolved in deionized water which was made from the Milli-Q1 reagent-grade water making systems (Millipore Corp., MA). Before successive application of an adapting and a test solution. the tongue was usually prc-adapted to a frog Ringer solution containing (mM) I Il.2 NaCI. 1.9 KCI. I.1 CaCI,. 0.1 NAH?PO, and 2.4 NaHCO,. adjusted to a pH of about 7.5.

When recording the whole glossopharyngeal nerve activities. the Ringer solution that was flowed continuously over the tongue was stopped during about 30sec before application of an adapting solution. but a thin layer of the Ringer stayed on the tongue surface. Application of adapting bitter solutions was alternated with test salt solutions without interruption of fluid flow. When recording the responses from taste units and cells, the tongue was exposed to a continuous flow of Ringer solution, which was terminated immediately when an adapting solution followed by a test solution was presented. In the experiments with the whole taste nerves and units. an interval of at least 3 min elapsed between each pair of the adapting and test stimulus. The rate of solution flowing from delivery nozzles of the gustatory stimulator was 0.362 ml/set for recordings from the whole taste nerve and 0.129 ml/set for recordings from taste units and cells. All the experiments were carried out at room temperature ranging from 22 to 26°C.

RESULTS

I. Enhanciny effect of 10 set Q-HCI adaptation on gustatory neural responses to various salts

Figure I shows the integrated whole glossopharyngeal nerve response to 0.1 M salts after the tongue was adapted to Ringer, water and 1 mM Q-HCI. As can be seen in the figure, the gustatory neural responses to the NH,+CI are mainly composed of an initial phasic response, but those to the CaCI, are composed of an initial phasic response followed by a smaller tonic one. The amplitude of the tonic re-

965

TOSHIHIIX SATO md KL;AfIh(l SKIMOTO

RI= WaterOlM imM NH&l NH&L Q-HCI NH&l

20 set

RlngerOIM coc1,

*z ImM C&l, Q-HCI CaC1,

Fig. 1. Changes in an initial phasic component of integrated gustatory neural responses to 0.1 M NH,CI (A) and 0.1 M CaCI, (B) after adaptation of the tongue to three kinds of solutions. Adapting solutions applied for IOsec: Ringer (Al. BI); deionized water (AZ. B2): and 1 mM Q-HCI (A3, B3). Stimulus salts: 0.1 M NH,CI (A); and 0.1 M CaCI, (B). Thin and heavy lines below the response denote application of an adapting and a test solution, respectively. In this and subsequent figures. the tongue was pre-adapted to a Ringer solution before application of the adapting solutions and it was rinsed with the Ringer after the test solutions. Data from two distinct preparations with different

amplifications.

NaCl LiCl KC1 NH&( KNO,

(3)

RWQ RWd RW 0

100 c

50 -

8

_ o-

1

I

0 2 .i?

I00

50 0 I

T

#I RWO

CaCl, WC h Ia2 i.

m! RWQ RWQ RWQ RWQ

Adaptmg solutions

Fig. 2. Amplitudes of initial phasic neural responses to various salts of 0.1 M after IOsec adaptation to Ringer (R). deionized water (W) and 1 mM Q-HCI (Q). The, response amplitude in ordinate is given relative to the response to a salt following 10 set of I mM Q-HCI adaptation Numerals in parentheses are the number of preparations used. Vertical bars show S.E. of the means. Abscissae

give three kinds of adapting solutions and test stimuli are shown above the parenthesis.

Enhancement of salt responses after adaptation to quinine 967

I00 A

s 5;d _ I,,], % 8 Id.0 -4 -3 -2 -I 0

NaCl , log M 6 L

0 5 2

100

5c

0

.D

.il v H,O -5 -2

CaCb, log M

I I I I I I 1 I I I -2 -I 0 -2 -I 0

KCi , log M NaHCO,, log M

.E

f l After ImM Q-I-ICI

0 After Ringer

Pi,” -3 -4 -5 -z -1 ”

NaH,PO,, log M

Fig. 3. Concentration-phasic neural response curves with NaCl (A). KC1 (B). NaHCOs (C). CaCl, (D) and NaH2P04 (E) different salts after IOsec adaptation to I mM Q-HCI (0) and Ringer (0). Data in (A) and (E) were obtained from two different preparations and data in (B). (C) and (D) were from the same preparation. In each graph, the maximum response after the Q-HCl adaptation

is expressed as 100’5;.

sponses to CaCl,, as well as to other salts, remained constant under the three kinds of adapting solutions, while the amplitude of phasic responses to CaCl, and NHJl was markedly augmented following Q-HCl adaptation (Fig. lA3 and B3). Figure 2 summarizes the amplitudes of initial phasic responses to nine salts of 0.1 M and deionized water after adaptation to Ringer, water and 1 mM Q-HCl. It is seen that the salt response amplitudes are generally arranged in the order of 1 mM Q-HCl > water > Ringer adaptation. The rate of salt response enhancement after Q-HCI was, in general, Iarger with monovalent salts than with divalent salts. No significant difference was found between the neural responses to deionized water after Ringer and 1 mM Q-HCl, indicating that the salt response enhancement was regardless of the water solvent in the salt solutions.

Figure 3 illustrates the concentration-neural response relations obtained by five salts after adaptation to Ringer and 1 mM Q-HCI. In the preparations with which these measurements were carried out, the so-called water response was very small. It is seen that the neural responses to the salts when applied after 1 mM Q-HCl were larger in a wide range of concentrations than those when applied after Ringer, and that the threshold concentration for salts after the Q-HCl became lower by l-3 logarithmic units.

2. Enhancing effects of adaptation to other bitter compounds on neural responses to salts

The effects of adaptation to five other bitter substances such as QH2S04, brucine, picric acid, caffeine and nicotine were examined. Figure 4(A) illustrates one example of these experiments where the enhanced responses to 0.1 M NH&l were obtained. As can be seen here, the initial phasic neural response to the NH4Cl was larger after 1 mM Q-HCl, Q-H2S04 and picric acid adaptation than after Ringer and water adaptation. The tonic response was hardly elicited by the application of 0.1 M NH.,Cl after bitter stimuli, except a small tonic response after 1 mM picric acid. Adaptation to 1 mM picric acid out of bitter compounds tested caused pronounced tonic responses to 0.1 M NaCl (B3), 0.1 M KC1 (C3) and other monovalent salts. which were hardly seen under Ringer adaptation. In the cases of CaC12 and MgCl, stimulation where a remarkable tonic response was produced even under Ringer and water adaptation, the amplitude of the tonic response was enhanced by picric acid adaptation but not by the other bitter substances.

Figure 5 gives the effects of adapting to various 1 mM bitter solutions for 10sec on initial phasic neural responses elicited by 0.1 M salts. The salt responses after 1 mM caffeine, nicotine and brucine

Ringer WGZGi ImM NH,Cl NH,CL Q-HCl NH, CL

ImM O.lM Q-H,SO, NH,CL

ImM OIM Pwlc NH,Cl acid

ImM OIM Bruclne NH,Cl

I

B-

Water 0.1 M NaCl

ImM OIM Q-HCI NaCL

I mM O.IM Picw NaCl acid

20 set

CA

WG KC1

4L

2

ImM Q-HCI KC1

ImM 0.1 M Picric KC1 acid

Fig. 3. Integrated neural responses to successive applications of various types of adapting solutions and test salt solutions. A thin line below each record denotes the duration of adaptation lo Ringer. water or bitter solution and heay line the duration of stimulation with 0.1 M NH,CI (A). NaCl (B) or KCI (Cl. In record C3 the response to 0.1 M KCI is truncated. Records (A). (B) and (C)

were obtained from different preparations.

were larger than those after Ringer adaptation but they were the same as or smaller than the salt responses after water adaptation except for the effect of brucine on a 0.1 M NaCl response. From adap tations to the six I mM bitter solutions. water and Ringer, the magnitude of initial phasic neural responses to salts was generally arranged in the following order: picric acid > Q-H,SO, 2 Q-HCI > bru- tine 2 water > nicotine = caffeine > Ringer.

3. Effect of concentration of adapting Q-HCI solution on gustatory nerve responses

Figure 6(A) illustrates the integrated whole nerve responses to 0.1 M MgCI, when the tonque was adapted to varying concentrations of Q-HCI for 10sec. It is seen that the initial phasic responses increase with increasing concentration of Q-HCI, whereas the tonic responses are independent of


A

0.1 M

Adapting

B OSM

MgCI,

a

D

O.IM NH,CI

Fig. 5. Histograms showing the amplitudes of initial phasic neural responses to 0. I M salts after 10 set adaptation to water, Ringer and various bitter solutions of 1 mM. In abscissae adapting solutions are shown and in ordinate the amplitude of initial phasic responses is expressed as percentage of the response to each salt after 1 mM Q-HCI. Test salts of 0.1 M were NaCl (A), MgCl, (B), KC1 (C) and NH,Cl (D). Hatched bars represent remarkable potentiation of salt responses after adaptation to 1 mM solutions of Q-HCI, Q-H2S0, and picric acid, compared to responses after Ringer and

water adaptation. Data from one preparation.

4c

B

A0.1 M MgC4

‘o_/ Fi 0 0 r”

lo-5 lo+ 1o-3 .c_ !x Q-HCL, M

A

-L-

Water MgC$

IOseC

_L

O,“!;,“, MgC$

Fig. 6. Change in the amplitude of initial phasic neural responses to salts as a function of concentrations of an adapting Q-HCl stimulus. (A) Integrated neural responses to 0.1 M MgC12 following 10sec adaptation to water and 0.01 mM-1 mM Q-HCl. (B) Relationships between the concent:ation (M) of an adapting Q-HCl solution applied for IOsec and the initial phasic neural response (arbitrary units) to 0.1 M MgCI,, NH,Cl and NaCI. The responses to the salts after Ringer adaptation are

plotted at the left. Data from one preparation.

970 TOSHIHIDE SATO and KUMIKO SUGIMOTO

A h-.-i -

ImM OIM I mM 0.1 M ImM OIM Q-HCl KC1 Q-Xl KC1 Q-HCl KC1

I mMTM I rnMTM ImM OIM Q-HCl NH4CL Q-HCL NH,CL Q-HCl NH,Cl

0 NaCl

” I” L”

Duration of ImM Q-HCl adaptation , set

Fig. 7. Change in salt response amplitude as a function of the duration of 1 mM Q-HCI adaptation. Integrated neural responses to (A) 0.1 M KC1 and (B) 0.1 M NH&I after adaptation to 1 mM Q-WI for 3%30sec. (C) Relation between the amplitudes of initial phasic responses to four types of 0.1 M salts and the durations of 1 mM QPHCl adaptation. At the right. the test solutions used and the response to each salt after Ringer adaptation are shown, The response amplitude in ordinate is expressed

in arbitrary units. All the data were from one preparation.

Q-HCI concentration. Figure 6(B) shows the relationships between the concentration of adapting Q-HCl solutions and the initial peak amplitude of whole nerve responses to 0.1 M solutions of MgCI,, NH+Cl and NaCl. It is clear that the amplitudes of initial phasic responses to the salts increased gradually depending on the Q-HCI concentration.

4. Effects of duration of Q-HCl adaptation on gustatory neural responses to salts

Short adaptation. In Fig. 7(A) and (B), an experiment is shown in which the initial phasic neural responses to 0.1 M KC1 and 0.1 M NH4Cl became larger as the duration of 1 mM Q-HCl adaptation was gradually increased up to 30sec. Figure 7(C) illustrates the relationships between the period of 1 mM QHCl adaptation and the amplitude of initial phasic neural responses to test stimuli of 0.1 M salts. With NaCl and KC1 the response increased gradually, whereas with NH&l and CaCl, the response reached

almost the maximum value 10 set after 1 mM Q-HCI was applied.

In Fig. 8(A) is shown a rapid return of an enhanced NaCl response induced by Q-HCl adaptation (Al and A2) to its control level (A3 and A4) when a water rinse for short periods was interposed between the end of 10sec adaptation to 1 mM Q-HCI and the onset of 0.3 M NaCl application. Figure 8(B) illustrates the relation between the amplitudes of initial phasic neural responses to 0.3 M NaCl and the durations of a water rinse following 10sec Q-HCl adaptation. It is seen that the enhancing effect of Q-HCl disappeared by about 5 set water rinsing of the tongue after the end of 10 set Q-HCl adaptation.

Long adaptation. To examine the effect of prolonged application of an adapting Q-HCI solution on a salt response, 1 mM Q-HCl was delivered continually to the tongue for 15 min and 0.3 M NaCl stimulus pulses of 3 set duration were given at a frequency of l/min by interrupting the Q-HCl flow for

Enhancement of salt responses after adaptation to quinine

A IO set v -

971

RQNa R QWNa R QWNa R WNo R

2 IOO-

2

6 80-

:: ’ 60-

B !i z 40-

%

jj 2o

I I I I I I 5 IO 15

Duration of water rinsing after IO set I mM Q-I-ICI adaptation, set

Fig. 8. Rapid disappearance of a salt response enhancement evoked by a short period of Q-HCl adaptation. (A) Change in integrated initial phasic neural responses (arrow heads) to 0.3 M NaCl stimuli with time after 10sec adaptation to 1 mM Q-Ha. A water rinse was interposed between the Q-HCl and the NaCI. Duration of a water rinsing: 0 set (Al); 3 set (A2); and 5 set (A3). Abbrevia- tions in the experimental procedure shown underneath the records: R, Ringer; Q, 1 mM QHCl; Na. 0.3 M NaCl; W, deionized water. (B) Relation between the amplitude of initial phasic neural responses [arrow heads in (A)] to 0.3 M NaCl after 10 set adaptation to 1 mM Q-HCI and the duration of a water rinse interposed between them. In ordinate, NaCl response without water rinse after Q-HCI

is taken as IOO’?. 0, response to 0.3 M NaCl after IOsec water adaptation alone as in A4.

each 3 set period. Figure 9 illustrates such an experiment. As shown in the curve obtained, the Q- HCI-induced enhancement of phasic NaCl response reached a maximum l-2 min after Q-HCl adaptation and then decreased gradually through the level of the response to 0.3 M NaCl following 10 set water adaptation. No enhancement was observed in the tonic response, which commenced to decrease at approx 3 min after the onset of Q-HCl adaptation and then decreased progressively with increasing Q-HCl adaptation time.

The inset B of Fig. 9 shows a procedure to evaluate the recovery of the depressed NaCl response after the end of a 15 min Q-HCI flow, where the Ringer solution was flowed continually and a water rinse was presented to the tongue for 10sec before application of a test 0.3 M NaCl solution every 5 min. As shown in the curve connecting the closed circles, the depressed phasic responses to 0.3 M NaCl recovered very slowly: an almost 30min period of Ringer flow was required to reach the control NaCl response level following 10sec water adaptation. The depressed tonic responses recovered by the similar time course.

5. E&t of transient mixing of uu adapting Q-HCl solution and a test salt solution on salt response enhancement

Since the amplitude of enhanced salt responses changed as a function of Q-HCl adaptation periods (Fig. 7). it is probable that the mechanism by which the salt response enhancement was brought about is due to the process of Q-HCl adaptation occurring on the taste receptor membrane. However, since in most of the foregoing experiments no rinse was given between cessation of Q-HCl adaptation and onset of salt application, there occurred a transient mixing of the both kinds of substances. Thus, there is a possibi- lity that a portion of the enhancement of an initial phasic response to a salt may originate from the mixing, if a stimulant action of one component in the mixture is enhanced in the presence of the other component or if both components of the mixture exert mutually the enhancing action. To examine this possi- bility the mixture containing various concentrations of Q-HCI and 0.1 M salt was applied as a test stimulus after the tongue had been adapted to 1 mM

972

a

5 01

0 It I I I I” I 1 I r

0 5 IO I5 25 35 45 55

Time. mln

Fig. 9. Change In neural response to 0.3 M NaCl during prolonged adaptation of the tongue to 1 mM Q-HCI and its recovery after returning to Ringer. O. change in initial phasic response amplitudea lo repetitive application of 0.3 M NaCl (3 set) at I,/min during 15 min adaptation to 1 mM Q~-HCI; 0. change in initial phasic response amplitudes to repetitive application of 0.3 M NaCl every 5 min after shifting the Q-HCI to Ringer adaptation. Responses to 0.3 M NaCl of open circles except for the first three were obtained by a procedure as shown in the inset A, and those of closed circles were obtained by a procedure of the inset B. where prior to application of 0.3 M NaCl the tongue was rinsed with deionized water for each 10 set by stopping temporarily the Ringer flow. Abbreviations in insets A and B: Q. 1 mM Q-~HCI: Na. 0.3 M NaCl: Rp. amplitude of initial phasic response. R. Ringer: W, deionized water. Point 1 (0) shows a response to 0.3 M NaCl after Ringer without Q-HCI adaptation and point 2 (0) a response to 0.3 M NaCl after IOsec water adaptation. the level of which is shown as a dotted line. Values of the first three open circles were obtained with separate measurements in which 0.3 M NaCl was applied after 2, IO or 30sec adaptation to I mM Q-HCI. Repetitive application of 0.3 M NaCl at l/min even under Ringer adaptation caused a very gradual reduction of the initial phasic response. so that open circles were corrected for the reduced ratios.

All the data were obtained from one preparation.

Q-HCI for IOsec. In Fig. 10, four series of such experiments are shown. Graph A illustrates the initial phasic response heights to various concentrations of Q-HCI alone and to the mixture of various concentrations of Q-HCI plus 0.1 M KCI. Application of water to 1 mM Q-HCI stimulus after a 10sec period of 1 mM Q-HCI adaptation did not cause any responses, and the amplitudes of responses to the mixtures after 1 mM Q-HCI were almost independent of Q-HCI concentration in the mixtures. It is also seen in graphs B-D that both the initial phasic responses and the following tonic responses to the mixtures of various concentrations of Q-HCI and 0.1 M salt after I mM Q-HCl adaptation are almost independent of Q-HCI concentrations mixed. indicating that only the 0.1 M salt (NH,CI, MgCI, or CaC12) in the mixture is associated with the generation of the salt responses. From these series of experiments it is obvious that a small amount of an adapting I mM Q-HCI solution that is transiently mixed with 0.1 M test salt, when the former is switched to the latter, is not contribu- table to the mechanism of salt response enhancement. In other words, the salt response potentiation after the adaptation of the tongue to Q-Ha and probably other bitter compounds is due to an adaptation process which may take place on the receptor membrane of taste cells.

6. Inhibition of potentiating action of Q-HCI solution by adding salt

In the foregoing experiments, all the bitter solutions

were made with deionized water and applied to the tongue as an adapting solution. Figure 11(A) is an example of neural responses showing the comparison between adaptation effects of Q-HCI dissolved in deionized water (A3) and in 0.1 M NaCl (A4). It is seen that the amplitude of initial phasic response to 0.1 M MgCI, is much larger after Q-HCI in water than after Q-HCI in 0.1 M NaCI. while the amplitude of the tonic responses remains relatively constant even after Q-HCI either in water or in 0.1 M NaCI. Figure 1 l(BHD) shows the histograms of initial phasic neural responses to three kinds of 0.1 M salts when adapted for 10sec to five adapting solutions. Only Q-HCl in water was effective in augmenting salt responses. When 1 mM Q-HCI in 0.1 M NaCl or Ringer was used as adapting stimuli. the gustatory neural response to a salt was similar to that after Ringer adaptation (Fig. 11C and D). These experiments clearly indicate that the ability of Q-HCI solution to enhance the salt response is suppressed by adding salts to it.

To understand precisely the relation between the salt concentration added to a @HCI solution and the degree of suppression of the potentiating Q-He action, 1 mM QHCl solutions containing various amounts of NaCI were delivered to the tongue as adapting solutions, and their effects on neural responses to test salt solutions were measured. In Fig. 12, these two experiments are illustrated. Figure 12(A) shows the amplitudes of initial phasic neural responses to the test stimulus of 0.1 M NaCl after


Test sdut’lon

l Q-HC 1+ 0.1 m KC1 I

4 d---O l - 20 Phaslc response

Phow response .-.-

+-.

---___-_-__ mal

n bl

t - Q-HCL, M ” Q-HCL, PL

5 zs c”

C

2 5 p 30

t

Phaw response

‘, 2

-N----e

0 k! 2

Tonic response

Test solutlon

.GHCLtO IM NH&L

AQ-HCL

Test xdutm D

l \Q-HCl

o i +OlM MgC$

A Q-HCL Phaswz response

al m

n bl

a2 0 Ob2 Tom response

Test solution

. 1 Q-HCL

0 j +OlM CA:;,

A Q-HCL

ma1

mbl

a2 0 ob2

I I 1 ,. I L

Ii,0 10-5 10-4 10-f -1

Hz0 lO-5 IO+ lO-3

Q-HCL, M Q-HCL, M

Fig. 10. Gustatory neural responses to mixtures of various concentrations of Q-HCI and 0.1 M salt (0. 0) and to Q-HCl alone (A) after 10sec adaptation of the tongue to 1 mM Q-HCI. Test solutions are shown at the upper part of right in each graph. In the mixtures containing Q-HCI and 0.1 M salt, the type of salts was (A) 0.1 M KCI, (B) 0.1 M NH&I, (C) 0.1 M MgC12 and (D) 0.1 M CaCI,. A dashed line in each graph shows the amplitude of neural response to application of I mM Q-Ha itself. n al and 0 a2. if present, show, respectively, the phasic response and the tonic response to 0.1 M salt after 10sec water adaptation. and W bl and 0 b2, if present. show. respectively. the phasic and the tonic response to 0.1 M salt under Ringer adaptation. Abscissae show Q-HCI concencentration which was mixed with 0.1 M salts when necessary. In ordinates, neural responses are expressed in

arbitrary units. Each graph was obtained from a different preparation with little water response.

the tongue was adapted for 10 set to various concentrations of NaCl alone and to the mixtures containing various concentrations of NaCl plus 1 mM Q-HCl. As shown in the curve C-I, the responses to test 0.1 M NaCl decreased gradually with increasing concentration of adapting NaCl solution. When the mixture of 1 mM Q-HCI and NaCl ((M.l M) was used as an adapting solution, 0.1 M NaCl response (C-2) was at a maximum under the adaptation to 1 mM Q-HCI in water, reduced gradually up to a 1 mM concentration of NaCl added to the Q-HCl, and then fell down rapidly up to 1OOmM NaCl. If this reduction of 0.1 M NaCl responses is due to simply to the difference in NaCl concentration between the adapting and

the test solution, it would be estimated that a reduction curve for the test stimulus of 0.1 M NaCl after adaptation to the mixture of 1 mM QHCl and NaCl may become like the curve C-3. However, the actually obtained curve C-2 indicates that the above simple estimation is not reasonable. Therefore, it is conceiv- able that a large reduction of 0.1 M NaCl responses following adaptation to 1 mM Q-HCl plus NaCl above 1 mM may be due not only to the NaCl concentration difference between the adapting and test solutions but also to a reduction of the enhancing action of Q-HCl molecules in the presence of NaCl.

Graph B in Fig. 12 also shows another experiment similar to the graph A except that the test solution

TOSHIHJDE SATO and KL:MJ~;O SLIGJMU~O

Ringer OIM Water OIM ImM OIM ImM OIM

MgCLz MgCLz Q-HCl MgCl, Q-HCl MgCl? In H,O InOlM _

NaCl

OIM ICI I

OIM KC1

OiM

MgCL,

Adopting solutions

Fig. 11. Depression of an enhancing action of Q-HCI on salt receptors by dissolving it in salt solution. (A) Integrated neural responses to 0.1 M MgCI, after a IOsec period of adaptation lo Ringer (1). deionized water (2). 1 mM Q-HCI in deionized water (3) and 1 mM Q-HCI in 0.1 M NaCl (4). (B). (C) and (D) Histograms showing the amplitudes of initial phasic responses to 0.1 M NaCl (B). 0.1 M KC1 (C) and 0.1 M MgClz (D) after IOsec adaptation to five kinds of adapting solutions (abscissae). The amplitudes of responses in ordinate are expressed in arbitrary units. All data were from one

preparation.

was 0.1 M MgCI,. In this figure, the amplitudes of neural responses to 0.1 M MgCI, are plotted for both the phasic and tonic components. Following adaptation to mixtures containing 1 mM Q-HCI and various concentration of NaCI, the rate of reduction of the phasic Gsponses (the curve C-2) was larger than an estimated curve C-3 that was similar to that in Fig. 12(A). The tonic response under NaCl adaptation was same as that under adaptation to the mixture.

7. EfSect of 1 mM Q-HCI adaptation upon gustatory unit responses to salts

As was described previously (Sato, 1976a. 1978). a fungiform papilla on the tongue surface was fully drawn into a recording suction electrode, and gustatory impulses conducted antidromically along the nerve fibers within the papilla were recorded during

applying an adapting solution of 1 mM Q-HCI and a test salt solution. The test salts employed for unit examination were NaCI, CaCIZ and NaH2POJ.

Figure 13 illustrates the dose-response curves for NaCl of five units obtained from five different! papillae. It is clearly seen that, excepting one unit (+. 0). the unit responses to NaCl after the Q-HCl were markedly augmented compared to those after Ringer, and that 0.1 M threshold concentration for NaCl under Ringer adaptation shifted to 0.0014 1 M under the Q-HCI adaptation. The enhancement pattern of gustatory units to the other salts applied after Q-HCI was similar to the NaCl response enhancement.

8. Response types of gustatory unitb to successive uppli- cation of adapting 1 mM Q-HC1 and test 0.5 M NaCl

Table 1 gives types of gustatory unit responses when only a pair of 0.5 M NaCl and 1 mM Q-HCl


n

G 25- 9

0

AdaDtlna solution I L? g 50

I

z 40

0

P 30

t

f 2o L

= IO

Fl i t 0

1)’ - I mM O-HCL t NaCl

NaCL concentration in adapting solution, M

Fig. 12. Depression of an enhancing action of 1 mM Q-HCI solution by adding various amounts of a salt to it. (A) Changes in initial phasic neural responses to a test solution of 0.1 M NaCl after IOsec adaptation to I mM Q-HCI solution containing various concentrations of NaCl (0) and to various concentrations of NaCl alone (A). (B) The amplitudes of neural responses to 0.1 M MgCl, after 10 set adaptation to mixtures of 1 mM Q-HCI plus various concentrations of NaCl(0, 0) and to various concentrations of NaCl alone (A, A). The magnitudes of initial phasic responses are shown in closed circles (the curve C-2) and closed triangles (the curve C-l), and tonic responses in open circles and open triangles. The curve C-3 (dashed line) in (A) and (B) shows an estimated curve when the rate of depression of phasic responses lo the test stimulus of 0.1 M NaCl or 0.1 M MgClz after adaptation to the mixtures was assumed to be only due to an effect of NaCl concentrations in the adapting mixtures. At the right in (A) and (B), the phasic and tonic responses to the salt following 10sec Ringer adaptation are shown. Data in (A) and (B) were obtained from two different

preparations.

were applied as taste stimuli. The gustatory units were classified in three types, and the response character- istics to 0.5 M NaCl after 1 mM Q-HCl adaptation were classified as listed in the fourth column of the table. Of course, the type 1 units did not respond to 0.5 M NaCl before and after Q-HCl adaptation. so that there was no subtype. In type 2 and 3 units. 0.5 M NaCl responses after 1 mM Q-HCl were char- acterized by enhancement. depression and no change compared with the control response under Ringer adaptation. They imply that impulse numbers elicited by 0.5 M NaCl after 1 mM Q-HCl were increased, decreased and unchanged, respectively. The enhancement was found in 667; of the type 2 units and in 67’4 of the type 3 units. The depression that was found in 12gb of the type 2 units and 24”;, of the type 3 units was generally weak. It is clear that the enhancement of the glossopharyngeal nerve responses elicited by NaCl and probably other salts after Q-HCl adaptation resulted from the “enhancement units” of the type 2 and 3 units.

9. Distribution ofgustarory units responding to Q-HCl and/or NaCl

Figure 14 illustrates the frequency distribution of peak-to-peak amplitudes (PI’) of impulses in the three types of units classified in Table 1. At the time of recording from the units, suction electrodes of only approx 2OOpm i.d. at the tip were used to suck the round shape of the fungiform papillae of about 3OOpm dia, so that it was possible to compare the spike amplitudes of individual units in spite of extra- cellular recordings. The type 1 units responding to Q-HCl alone had on the average the smallest amplitude (see Fig. 14). There was no significant difference in spike amplitude between the type 2 and 3 units. However, a highly significant difference (P < O.OOl) was found between type 1 and 2 units and between the type 1 and 3 units. In type 2 units. those units which indicated enhancement, depression and no change of 0.5 M NaCl responses after Q-HCl had the spike amplitude of 64 + 3 @V (n = 35). 62 + 3 PV (n = 12) and 69 f 13 PV (n = 4), respectively. Simi- larly. in the type 3 units, the units showing enhancement, depression and no change of the NaCl responses had the spike amplitude of 54 + 3 PV (n=35),67+6~V(n=7)and69fll~V(n=lO). respectively. There was no significant difference between spike amplitudes in the enhancement units and the other subtype units.

Table I. Classification of gustatory units from the responses to I mM Q-HCI and 0.5 M NaCl

Types of units No. of

observations Y0 Response to 0.5 M

NaCl after 1 mM Q-HCI No. of

observations “0*

Type I : Q-HCI-sensitive and NaCl-insensitive units

Type 2: Q-HCI-insensitive and NaCl-sensitive units

Type 3: Q-HCI-sensitive and NaCl-sensitive units

36 26

51 37

52 31

Nothing Enhancement Depression No change Enhancement Depression No change

36 100 35 66 I2 24 4 IO

35 67 I I3

IO 20

* Percentage in each type.

z 40-

b f 20-

: 0-

After I mM Q-HCI After Ringer

I I I I I I I 1 I I

001 0.1 1.0 0 01 0.1 IO

NaCl, M NaCI. M

Fig. 13. Number of impulses of five gustatory units discharged in 5 set after application of various concentrations of NaCl following IOsec adaptation to I mM Q-HCI (A) and Ringer (B). A pair of symbols in similar shapes such as open and closed circles show responses from the same unit. Each

unit belonged lo the type 3 unit in Table I and was obtained from different fungiform papillae.

The conduction velocities corresponding to the spike amplitudes were, with several fungiform papillae, determined by electrically stimulating the whole glassopharyngeal nerve at the point about 5 cm dis- tant from the proximal end of the tongue and by recording antidromic action potentials from the axons within a single fungiform papilla, As can be seen in Fig. 14. the mean conduction velocities of the types 1. 2 and 3 units were 7.1. 9.8 and 10.8 m/set. respectively. Axon diameters were calculated on the basis of the formula by Tasaki (1953). using the conduction velocities measured. The type 1 gustatory units which respond to Q-HCl alone had the slowest conduction velocity and thereby the smallest axon diameter.

10. Intracellular recordings of tuste cell responses to salts following I mM Q-HCl adaptation

Sato (1977, 1978) has shown that the magnitude of an initial phasic depolarization in taste cells varies depending on the rate of rise of a taste stimulus. Therefore, a microelectrode was inserted into a taste cell within the fungiform papilla which was located near injection needles that served as delivery nozzles of the gustatory stimulator. Figure 15 illustrates three examples of depolarizing receptor potentials of taste cells to three salts following Ringer adaptation and 1 mM Q-HCl adaptation. In the left records Al-Cl. only sustained depolarizations were initiated by salt stimuli following Ringer adaptation, while in the right records AZ-C2, depolarizations to the salts following 1Osec of 1 mM Q-HCl adaptation comprised an initial phasic response followed by a steady-state response. The rate of rise of a depolarization to salt stimuli was more rapid under Q-HCl than that under Ringer. Application of the adapting solution of 1 mM Q-HCl in deionized water gave rise to either depolarizing or hyperpolarizing potentials in taste cells. In the case of three cells of Fig. 15. application of the

adapting Q-HCl solution produced a hyperpolarizing response alone, The amplitude of sustained depolarizations to salts after the Q-HCl adaptation was the same as that after Ringer adaptation.

11. Effect of duration of 1 rnM Q-HCI adaptation ml taste cell response

Figure 16(AHC) illustrates the change in intracellular responses in a taste cell to 0.1 M NH,Cl after the cell was adapted to Ringer and 1 mM Q-HCl. This cell showed a hyperpolarization during the

.Q-HCl adaptation whose amplitude gradually reduced in the depolarizing direction.

In Fig. 16(D). the depolarization amplitudes measured from the hyperpolarized level to the peak of depolarization (0 in B) and those measured from the resting potential level to the peak of depolarization (0 in B) are plotted against adaptation periods of 1 mM Q-HCI. It is seen that the values of filled circles (right ordinate) decrease with increasing period of Q-HCl adaptation, whereas the values of open circles (left ordinate) increase with increasing adaptation time. In the case of these open circles. the dependence of the initial phasic depolarization on the duration of the previously given Q-HCl adaptation was consis- tent with the initial phasic gustatory neural response to a salt after Q-HCl, as shown in Fig. 7.

When an adapting Q-HCl solution of I mM initiated a depolarizing response in a taste cell, the initial phasic depolarization elicited by a test salt stimulus superimposed on it and increased in magnitude with increasing duration of the Q-HCl adaptation. Therefore, as has been stated (Sato. 1978), it seems relevant to say that both the amplitude and the rate of rise of initial phasic depolarization to a salt in taste cells which arises from Q-HCl-induced depolarization level or resting potential level are the cause of the enhancement of gustatory neural responses to the salt after Q-HCl.


n-36

mean = 4223 PV

971

mean-5823 PV

1 40 60 00 100 120 140

Sprke amplitude , PV

I I 1 I 5 IO 15 20

Conduction velocity , m/set

I 1 1 I 25 50 75 100

Axon dlometer , pm

Fig. 14. Distribution of spike amplitudes of three types of gustatory units classified by application of 1 mM Q-HCI and 0.5 M NaCl under Ringer adaptation. (A) Type 1 units responding to only 1 mM Q-HCI out of the two stimuli used. (B) Type 2 units responding to 0.5 M NaCl alone. (C) Type 3 units responding to both I mM Q-HCI and 0.5 M NaCI. The ordinate shows the number of units observed. The upper abscissa shows the peak-to-peak amplitude of spikes in PV, the middle abscissa the conduction velocity of units with the above spike amplitude, and the lower abscissa the axon diameter of units calculated from the cotiduction velocity with Tasaki’s formula. Three arrows denote

the mean spike amplitude.

DISCUSSION solution to enhance salt responses is dependent upon its stimulant action per se.

The present results reveal that of six adapting bitter Since pharmacological studies on the antimalarial substances of Q-Ha, Q-H$O.,, picric acid, brucine, action of quinine and related alkaloids, a great caffeine and nicotine, the first three are markedly number of investigations indicate that quinine has a effective in producing an enhancement of frog gusta- dual action on the majority of unicellular organisms tory neural responses to salts. Stimulation of the ton- and tissues, i.e. (1) the initial stimulant action and gue with Q-HCl, Q-H2S04 and picric acid gives rise (2) the subsequent depressant or anesthetic action to a large gustatory neural response, while stimu- (Sollmann, 1957; Roll, 1970). A brief period of adap- lation with brucine, caffeine and nicotine produces tation of the frog tongue to Q-HCl or other bitter a smaller response. Therefore, as pointed out pre- substances induces an augmentation of gustatory viously (Sato, 1978) the ability of an adapting bitter neural responses to variety of salts (Figs. l-5). Similar

976

Al

- A+-; OIM ImM OIM KC1 Q-HCL KC1

OIM

MgCt,

I a ImM OIM

Q-HCl MgC1,

Cl c2

- -7-P

OIM ImM OIM NH,Cl Q-HCL NH4CL

20 set

Fig. IS. Intracellularly recorded taste cell responses to 0.1 M KCI (A), 0.1 M MgCI, (B) or 0.1 M NH,CI (C) after 10s~ adaptation to Ringer (I) and I mM Q--HCI (2). Each of the paired recordings was from a different taste cell. Resting potentials under Ringer adaptation were -41 mV (A). -22mV (B) and - 19 mV (C). Excepting horizontal bars marking the duration of solution applications. the

Ringer’s solution flowed continuously over the tongue.

enhancing effects of QHCI adaptation on a gustatory neural response to NaCl have already been demonstrated in rat (Zotterman. 1956). hamster (Yinon & Erickson, 1970) and frog (Sato, 1976b. 1978). Smith & Frank (1972) were not able to find the enhancement of initial phasic gustatory responses to salts even after the rat togue had been adapted to Q-HCI for 10 sec.

On the other hand, in agreement with the experiments by Zotterman (1949) and Akaike 8c Sato (1976) with frogs. the present observation demonstrates that a prolonged period of adaptation to Q-HCI causes rather a depression of the frog gustatory neural responses to salts (Fig. 8). These findings clearly support the idea that Q-HCl and possibly other bitter substances exhibit a dual action, enhancing and depressing. on the gustatory receptors, depending on adaptation periods.

The enhancement or depression of salt responses after adaptation of the frog tongue to Q-HCl may

be caused by the following three possibilities: (1) its action on the outer surface of the taste receptor membrane; (2) its action on the inside of the taste cell membrane; (3) its action on the inside of the taste cell through the taste receptor membrane and/or the cell membrane. At present, no direct evidence is presented to explain the mechanism underlying the changes of salt responses after Q-Ha.

The present results indicate that even a 3 set period of adaptation to Q-HCl can give rise to the salt response enhancement (Figs 7 and 9) and that an about 5 set water rinsing of the tongue surface after adapted to Q-HCl for 1Osec is sufficient to remove the enhancing action of Q-HCl on salt receptor (Fig. 8). It has been generally accepted that taste stimuli penetrate very slowly the gustatory epithelium (Beidler, 1967; Mistretta, 1971; Ozeki & Noma, 1972), except recent work by Hayashi (1978) who has found a relatively rapid penetration of some substances into the frog gustatory epithelium. Therefore. it is very likely


OL 1 I I I IO

0 IO 20 30

Duration of ImM Q-HCL adaptotion , set

979

v OIM ImM

.Kec ImM OIM

NH&l Q-K1 NH&l Q-HCI NH&L

Fig. 16. Intracellular responses of a taste cell to 0.1 M NH&I after various durations of 1 mM Q-HCI adaptation. (A) Control 0.1 M NH,&1 response after adaptation to Ringer. (B) and (C) 0.1 M NH,CI responses after IOsec (B) or 30sec (C) adaptation to the PHCl. (D) Changes in the amplitude of taste cell responses to 0.1 M NH,CI plotted as a function of the duration of Q-HCI adaptation. Left ordinate (0) denotes the amplitude of depolarizations measured as the value of the open circle in (B), i.e. the amplitude from the resting potential level under Ringer to the peak of the depolarization, and right ordinate (0) the amplitude measured as the value of the closed circle in (B), i.e. the amplitude from the hyperpolarized membrane potential level at the end of Q-HCI application to the peak of depolarization. Notice the appearance of an initial phasic depolarization after Q-HCI adaptation

(B and C).

that Q-HCI and other bitter substances may first act on the outer surface of the taste receptor membrane, leading to the salt response enhancement. This assumption is strengthened by the following finding and its possible explanation. That is, while short adaptation to Q-HCl in water causes the enhancing effect on a salt response, the adaptation to Q-HCl in 0.1 M NaCl or Ringer does not (Figs 11 and 12). In this case, the application of Q-HCl stimuli either in water or in saline brings about similar amplitudes to their own gustatory responses (Figs 10 and llA), so that the extinction of the enhancing action of Q-HCl in saline is not due to a reduction of the amount of Q-HCl molecules reaching the taste recep tor membrane. If the taste receptor membrane is easily permeable to Q-HCl, as has been suggested in the skeletal muscle membrane (Isaacson & San- dow, 1967), it is assumed that Q-HCl molecules either in water or in Ringer would penetrate equally into the taste receptor membrane and further into the taste cell, and would eventually produce a similar for-m of salt responses. However, the actual results mentioned above did not agree with this assumption. Therefore, as has already been suggested (Sato, 1978), we propose that rapid penetration of Q-HCl into the taste

receptor membrane and taste cell may not occur and that the salt response enhancement after Q-HCl may result from its action on the outer surface of taste receptor membrane.

Perhaps the short-term adaptation of tongue to Q-HCl and other bitters will induce a positive conformational change of the salt binding site on the taste receptor membrane, which facilitates an interaction of salt receptor site with salt stimulus. Sato (1978) has proposed that enhancement of neural response to NaCl after Q-HCl is associated with salt receptor sites activated during Q-HCl adaptation, which make activated complexes with NaCl stimulus. This mechanism would be applicable to the enhancement of responses in the taste nerve and cell to a variety of salts following Q-HCl adaptation. As shown in the results, this enhancement is mostly restricted in the initial phasic component (the phasic discharge in taste nerve and the phasic depolarization in taste cell), remaining the tonic component constant (Figs 1, 4, 8, 11, 15 and 16). Thus it is likely that a rapid interaction between salt stimuli and activated salt receptor sites may result in the appearance of an initial phasic depolarization in taste cells, which induces the enhancement of the initial.phasic discharge of gustatory

9x0 TOSHIHIIE SATO and KVMI~CO SUGIMOT-o

neural impulses. Constancy of the tonic response in the taste nerve and cell may suggest that there is no change of the number of salt receptor sites available to a salt stimulus before and after taste cells are adapted to Q-HCI.

On the other hand. adaptation to picric acid enhances both phasicand tonic neural response (Fig. 4). This may be due to the ability of picric acid to adsorb very strongly on the surface of the taste receptor membrane. thus leading to a strong modification of the salt receptor sites. Evidence is presented that there occurs a strong modification of the phasic and tonic components of gustatory neural responses to salts. when the frog tongue is exposed to some drugs (Kashiwagura et al.. 1977).

In contrast to the enhancing action of Q-HCI when adapted for a short period of time. the experiment (Fig. 9) on prolonged adaptation to Q-HCl in water shows that a salt response is remarkably depressed as compared with the response under water adaptation and that the depressed salt response recovers very slowly. Comparable finding has been obtained by Akaike & Sato (1976). who used I mM Q-HCl in IOmM NaCl as an adaptmg bitter solution. They could hardly find salt response enhancement even after adaptation of the tongue to the Q-HCI for a relatively short time. As shown in Figs II and 12 the discrepancy between their result and ours during adaptation to Q-HCI for short periods is due to the use of different solvents in Q-HCI solution.

In parallel with the depressant action of Q-HCl. Akaike & Sato (1976) have found a gradual decrease of membrane potential and a gradual increase of membrane resistance in the frog taste cells. A similar decline of the action potential and resting potential during a lengthy treatment with Q-HCl or Q-H2SOI is reported in skeletal muscle fibres (Falk. 1961; Hud- dart. 1971). Since quinine and its isomer quinidine are found to inhibit the potassium permeability of the cell membrane in the red blood cells (Armand- Hardy et ~1.. 1975) and photoreceptors (Hanani & Shaw. 1977). the gradual decrease of the taste cell membrane potential during prolonged application of Q-HCI is likely to be due to a decrease in potassium conductance. as has already been suggested by Akaike & Sato (1976). Further, they have assumed that a gradual depression of frog gustatory neural responses, including a salt response during long Q-HCI adaptation, may be connected with a slowly progressing penetration of the Q-HCl stimulus into the non-polar region of the lipid layer of the taste receptor membrane. From the present results. we agree with this assumption. and, in addition. we assume that a Q-HCI stimulus may penetrate gradually inside the taste cell.

Koyama & Kurihara (1972) and Kurihara et d. (1972) have proposed that salty taste is induced by the binding of salt stimuli to the polar region of lipids in the gustatory receptor membrane and that bitter taste is induced by the penetration of bitter stimuli into the non-polar region of the membrane lipid layers. Since the gradual penetration of Q-HCl molecules into the taste receptor membrane is very likely to be related to rather inactivation process of taste cells. presumably taste receptor stimulation by a bitter substance is induced by a rapid interaction of the

bitter stimulus with a hydrophobic portion of lipids of the taste receptor membrane which is very close to the polar portion.

If Q-HCI molecules are not easily able to penetrate deeply inside the lipid layer of taste receptor membrane and exert. for long periods. only enhancing action on salt receptor sites of the outer surface of receptor membrane. the curve in Fig. 9. which shows the time course of gustatory neural response to a salt during prolonged Q--HCI adaptation. is likely not to fall down gradually but to be maintained at the maximum level of enhanced effect appearing approx 2 min after Q-HCl adaptation. Probably the gradual decline in the actual result is due to a balance between the enhancing action of Q-HCl on salt receptor sites and its depressing action on activity of the non-polar region of lipids in the taste receptor membrane.

In conclusion, we propose that the enhancing effect of Q-HCl on a salt response is due to its action on the outer surface of taste receptor membrane. while the depressing effect is due to the slou penetration of Q -HCI on the interior of the taste receptor membrane and taste cell.

Sl’MMAR1

I. An initial phasic component of the frog gustatory neural responses to 0.1 M salt solutions such as NaCI, KCI. NHLCl. CaCI, and MgCI, after a 10 set period of adaptation to 1 mM Q-HCl was greatly enhanced in amplitude compared with that after Ringer adaptation, but a tonic component of salt responses was not affected after the Q-HCl adaptation.

2. Adaptation of the tongue to Q-H$04 and picric acid produced a similar pronounced enhancement of the neural responses to salts, whereas the enhancing effects of brucine, caffeine and nicotine on the salt responses were very weak.

3. Potentiating effect of Q-HCI on a salt response was dependent on the adaptation periods and concentrations.

4. Potentiating action of a Q-HCI solution was suppressed by adding salts to it.

5. Analysis of the neural responses of gustatory units elicited by a pair of 0.5 M NaCl and 1 mM QHCI demonstrated that out of 103 investigated units responding to both NaCl and Q-HCI or NaCl alone. the 679; exhibited a marked enhancement of the NaCl response after the Q-HCI adaptation and the remaining 337; a suppression or no alternation of the NaCl response.

6. Intracellular recordings of taste cell responses to salts after 1 mM Q-HCI adaptation showed the appearance of an initial phasic depolarization which was not observed under Ringer adaptation. This phasic depolarization may be concerned with the enhancement of the initial phasic neural response to salts after QHCI. The magnitude of a steady-state depolarization following an initial phasic one to a salt stimulus was not changed after Q-HCI adaptation.

7. It is concluded that adaptation of frog taste cells to Q-HCI causes a conformational change of the outer surface of the taste receptor membrane which leads to the salt response enhancement in the taste cell and nerve following Q-HCI adaptation.

Acknowledgements-We thank Professor M. Ichioka olfactory receptor membrane. In Olfuction and Taste (Department of Physiology, Tokyo Medical and Dental Vol. 4 (Edited by SCHEIDER D.). pp. 234240. Wissen- University) for his continuous encouragement during the schaftliche Verlagsgesellschaft MBH, Stuttgart. present experiments and Miss M. Oguro for her help in MISTRETTA C. hj. (1971) Permeability of tongue epithelium preparing the manuscript. and its relation to taste. Am. J. Physiol. 220, 1162-l 167.

This study was supported in part by a grant (No. OZEKI M. & NOMA A. (1972) The actions of tetrodotoxin, 157425) from the Ministry of Education of Japan. procaine, and acetylcholine on gustatory receptors in

frog and rat. Jap. j. Physiol. 22. 467-475: ROLLO I. M. (1970) Drugs used in chemotheranv of

1 . I

REFERENCES malaria. In The Pharmacological Basis of Therapeutics (Edited by GOODMAN L. S. & GILMAN A.). 4th edition.

AKAIKE N. & SATO M. (1976) Mechanism of action of some pp. 1095-l 124. Macmillan, London. bitter-tasting compounds on frog taste cells. Jap. J. Phy- SA~O T. (1972) Adaptation of primary gustatory nerve re- siol. 26, 2940. sponses in the frog. Comp. iiochem. Physiol.-43A. l-12.

ARMANDO-HARDY M., ELLORY J. C., FERREIRA H. G., SATO T. (1974) Enhancement of salt resnonses in the froe FLEMINGER S. & LEW V. L. (1975) Inhibition of the cal- gustatdry nerve after adaptation to qiinine. Jap, J. ora; cium-induced increase in the potassium permeability of Biol. 16. 537. human red blood cells by quinine. J. Physiol., Land. 250, SATO T. (1975) Enhancement of gustatory neural response 32-33P. to salts following adaptation of frog tongue to quinine-

BEIULER L. M. (1967) Anion influences on taste receptor HCl. Tohoku J. exp. Med. 117, 381-384. response. In Olfaction and Taste, Vol. 2 (Edited by SATO T. (1976a) Latency of taste nerve signals in frog (Rana HAYASHI T.), pp. 509-534. Pergamon Press, Oxford. catesbeianal Experientia 32. 877-879.

FALI( G. (1961) Electrical activity of skeletal muscle. In SA~O T. (1976b) %-response of gustatory nerve following Biophysics of Physiological and Pharmacological Actions termination of quinine stimuli applied to the frog ton- (Edited by SHANES A. M.), pp. 259-279. American As- gue. Chern. Senses Flav. 2, 63-69. sociation for the Advancement of Science, Washington, SATO T. (1977) An initial phasic depolarization exists in D.C. the receptor potential of taste cells. Experientia 33.

HANANI M. & SHAW C. (1977) A potassium contribution 1165-l 167. to the response of the barnacle photoreceptor. J. Phy SATO T. (1978) Off-response in frog taste nerve and cell siol.. Lond. 270, 151-163. after stimulation of the tongue with bitter solutions.

HAYASHI H. (1978) Rapid penetration of potassium and Conlp. Biochrm. Physiol. 61A. 339-353. other salts into the frog tongue papilla. Jap. J. Physiol. SATO T. & SUCIMOTO K. (1977) Enhancement and inhibi- 28, 33-45. tion of gustatory responses after adaptation of frog ton-

HUDDART H. (1971) The effect of quinine on tension devel- gue to bitter-tasting compounds. 1; Food Intake and opment. membrane potentials and excitation-contraction Chemical Senses (Edited bv KATSUKI Y.. SATO M.. TAK- coupling of crab skeletal muscle fibres. J. Physiol., Lond. AGI S. F. & OOMURA, Y.j, pp. 163-171. University of 216, 64-657. Tokyo Press & Japan Scieniific Societies Press, Tokyo.

ISAACGON A. & SANWW A. (1967) Quinine and caffeine SMITH D. V. & FRANK M. (1972) Cross adantation between effects on 45Ca movements in frog sartorius muscle. J. salts in the chorda tympani’ nerve of the rat. Physiol. yen. Physiol. SO, 2109-2128. Behav. 8, 2 13-220.

KASHIWAGURA T.. KAMO N.. KURIHARA K. & KOBATAKE SOLLMANN T. (1957) A Manual of Pharmacology, 8th edi- Y. (1977) Enhancement of salt responses in frog gustatory nerve by removal of Ca” from the receptor mem-

tion, pp. 696-722. W. B. Saunders, Philadelphia. TA~AKI 1. (1953) Nervous Transmission. pp. 121-125. C. C.

brane treated with 1-anilinonaphthalene-8-sulfonate. J. Thomas, Springfield. Mem. Biol. 35. 205-217. YINON U. & ERICKSON R. P. (1970) Adaptation and the

KOYAMA N. & KURIHARA K. (1972) Mechanism of bitter neural code for taste. Brain Res. 23. 428432. taste preception: interaction of bitter compounds with ZOTTERMAN Y. (1949) The response of the frog’s taste fibres monolayers of lipids from bovine circumvallate papillae. to the application of pure water. Acta physiol. stand. Biochim. biophys. Acta 288, 22-26. 18, 181-189.

KURIHARA K.. KOYAMA N. & KURIHARA Y. (1972) Chemi- ZOTTERMAN Y. (1956) Species differences in the water taste. cal architecture and model system of gustatory and Acta physiol. stand. 37. 6@70.


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