J. Physiol. (1976), 254, pp. 87-107 With 10 text-ftgure8 Printed in Great Britain

87

ELECTRICAL RESPONSES OF FROG TASTE CELLS TO CHEMICAL STIMULI

BY N. AKAIKE, A. NOMA AND M. SATO From the Department of Physiology, Kumamoto University Medical School, Kumamoto, Japan (Received 7 March 1975) SUMMARY

1. Cells inside a fungiform papilla of the frog tongue were impaled with a glass capillary micro-electrode filled with 3 M-KCl. Cells considered to be taste cells showed a resting potential of about -35 mV and an input resistance of 17 MQ on the average. 2. Application of chemical stimuli such as salts, acids and quinine produced a sustained depolarization in a taste cell, the magnitude of depolarization being dependent on the stimulus concentration. Water and weak NaCl solution yielded a hyperpolarization. The thresholds for depolarization as well as the concentration-response relationships for various chemical stimuli in taste cells are in approximate agreement with those determined from the glossopharyngeal nerve responses. 3. The magnitude of depolarization produced by 0-1 M-NaCl and 003 M-CaCl2 was dependent on the membrane potential level and reduced linearly with a rise in the latter. However, depolarizations generated by 0001 M-HCl and 002 M quinine changed little in magnitude by a membrane potential change over a wide range. 4. During depolarizations induced by NaCl and KCl a marked reduction in the input resistance of a cell was observed, the amount of the reduction depending on the stimulus concentration. The reduction was also produced by CaC12 and HCl, but it is small compared with those by NaCl and KCl. Quinine produced an increase in the resistance associated with a depolarization. Water and weak NaCl solution produced an increase in the resistance associated with hyperpolarization. 5. The receptive mechanisms for various kinds of chemical stimuli are discussed in relation to changes in the membrane potential and the membrane conductance of taste cells.

88

N. AKAIKE, A. NOMA AND M. SATO INTRODUCTION

Slow depolarizations in response to various chemical stimuli have been recorded with a micro-electrode thrust inside a single taste cell of a fungiform papilla of the rat and hamster (Kimura & Beidler, 1961; Ozeki, 1970, f971; Ozeki & Sato, 1972) and of the frog (Sato, 1969, 1972). The depolarization varies in magnitude with variation in stimulus quality and concentration, and also from one cell to another. In addition, the results of these studies indicate that the cell is responsive to one or more of the stimuli representing the four taste qualities and is not specialized to be sensitive exclusively to but one type of chemical stimulus. Because of the graded properties the depolarizations have been considered to be receptor potentials in taste cells, which initiate impulses at the afferent nerve fibre terminal. Since the potential changes recorded from taste cells are considered to result from the preceding physico-chemical event between a chemical stimulus and the receptor membrane, a detailed study of changes in the membrane potential and in the membrane resistance of frog taste cells during chemical stimulations was carried out to elucidate the response properties of a taste cell and to throw light on the mechanism of chemoreception at the taste cell membrane. In addition, in order to examine how the membrane conductance varies with variation of adapting solution, the input resistance of taste cells adapted to various solutions was determined by measuring the current-voltage relationships in taste cells. Furthermore, variation in magnitude of the receptor potential produced by a given stimulus with change in the membrane potential was examined. A preliminary report of this work has already appeared elsewhere (Akaike, Noma & Sato, 1973). METHODS

All the experiments were carried out using bullfrogs (Rana cate8beiana) at room temperature of about 17° C throughout a year. The animal was anaesthetized by i.P. injection of 20 % urethane solution. To prevent the muscular movements of the tongue, the hypoglossal nerves and the geniohyoideus muscles of both sides were cut. The tongue was pulled out and its anterior portion was fixed with pins to the bottom of a Perspex chamber. The chamber had an inlet and an outlet, through which a solution (usually 001 M-NaCl) for adapting the tongue flowed throughout an experiment (Fig. 1A). Fungiform papillae at the anterior half of the tongue were studied in most of the experiments (Fig. 1 C). Stimulation of a papilla. In order to stimulate chemically a single fungiform papilla a test solution was applied through a thin polyethylene tube with about 50 jam hole diameter at its tip, which was placed close to a papilla to be tested (m.p. in Fig. 1). The other end of the tube was connected by another polyethylene tube to an injection syringe filled with a test solution (t.s. in Fig. 1). The test solution was ejected approximately at a flow rate of 1-5 ml./10 min under a hydrostatic pressure

FROG TASTE CELL RESPONSE

89

of about 40 cm H20 for a period varying from 10 to 60 sec. Stimulation of a fungiform papilla was stopped by moving the tip of the polyethylene tube away from the papilla by sliding the lucite holder of a tube with a micromanipulator. During the intervals between stimulations, the papilla was constantly rinsed with the adapting solution. In most experiments four polyethylene tubes, each of which was connected to a different syringe containing a different test solution, were employed (Fig. 1A), and consequently it was possible to stimulate a papilla consecutively four times with different test solutions. A

B

a.s.

C

t.t.

L

Fig. 1. Schematic diagrams of the experimental set up. A, general scheme; B, diagram showing position of the micro-electrode tip for impaling a fungiform papilla (f.p.) and micropipettes for chemical stimulation; C, portion of the tongue where the micro-electrode was impaled (t.t.). a.s., adapting solution; t.s., test solutions; c.e., calomel electrode; i.e., indifferent electrode; m.e., micro-electrode; m.p., micropipette; l.h., Lucite holder; s.p., screen plate for avoiding surface movement of fluid; p.b., paraffin block; l.p., Lucite plate; L, light source.

Recording the membrane potential. The membrane potential of a single cell was recorded with conventional micro-electrode techniques on an ink-writing recorder. Glass capillary micro-electrodes filled with 3 M-KCl and having a resistance of 2050 MW were employed for the experiments while the indifferent electrode was a 3 M-KCl-agar electrode placed in the adapting solution in the chamber (i.e. in Fig. 1). Both electrodes were connected through salt bridges (polyethylene tubes of

90

N. AKAIKE, A. NOMA AND M. SATO

about 1-3 mm internal diameter filled with 3 M-KCl-agar) to the calomel electrodes. They were led to a Wheatstone bridge circuit to record the potential change from inside a cell as well as to inject current pulses into the cell (Frank & Becker, 1964; Ozeki, 1971; Ozeki & Sato, 1972). The micro-electrode was advanced with a micromanuplator into a papilla through the centre of the sensory disk of a fungiform papilla (Fig. 1 B) under a binocular microscope until a steady negative potential of 20-30 mV in magnitude appeared suddenly. Chemical sensitivity of taste cells was examined by applying consecutively four kinds of test solutions to a fungiform papilla, into which a micro-electrode had been inserted. During the internals between successive stimulations the papilla was rinsed with 0-01 M-NaCl. Determination of electrical properties of cells. In many experiments hyperpolarizing current pulses of 5 x 10-10 A and of 100 msec duration were passed at a rate of once per second through a micro-electrode inserted inside a taste cell, and the resulting electrotonic potentials were recorded with the same electrode. The electrotonic potentials appeared suddenly after penetration of a taste cell with a micro-electrode. The magnitude of the electrotonic potentials often changed during chemical stimulation of the taste cell. The change in the magnitude which represents change in the input resistance of a cell was expressed relative to the value during the resting state of the cell. To indicate the relative magnitude of the membrane conductance during stimulation, the term 'relative conductance' which is the ratio of the electrotonic potential magnitudes between the rested and the stimulated state, was sometimes used in the present paper. Variation in the input resistance of a cell with a change in the membrane potential was measured by applying repetitive short hyperpolarizing pulses to the cell during passage of a very long pulse of either direction. For determining the current-voltage (I-V) relation, electrotonic potentials produced by injection of hyperpolarizing and depolarizing currents of varying intensities into a taste cell through a micro-electrode, which had been inserted in the cell, were recorded with the same micro-electrode. This procedure was repeated successively on several cells, adapted to 0-01 M-NaCl. Next, the adapting solution was changed to either one of 0-1 M-NaCl, 0-001 M-NaCl, 0-03 M-CaCl2, 0-02 M quinine hydrochloride and 0-001 M-HCl, and experiments similar to those described above were repeated on several taste cells. To measure a change in magnitude of the receptor potential with change in the membrane potential, the receptor potentials produced by one of 0-1 M-NaCl, 0-03 MCaC12, 0-02 M quinine hydrochloride and 0-001 M-HCl were recorded at varying levels of the membrane potential, the variation of the latter being performed by applying constant currents of varying intensities into a cell through the recording micro-electrode. Glossoph7ryngeal nerve response. In some experiments responses of chemoreceptors of the frog tongue to chemical stimuli of series of concentrations were recorded from the glossopharyngeal nerve using an integrator circuit having a time constant of 0-5 sec (Beidler, 1953). Test 80lution. Test stimuli employed were mainly HCR, quinine-HCl, sucrose, NaCl, KCl, CaCl2 and MgCl2. The former three were, in general, dissolved in 0-01 M-NaCl solution, while the latter four in deionized water. When experiments were carried out on taste cells adapted to Ringer solution, HCl, quinine hydrochloride and sucrose were dissolved in Ringer solution. The composition (mM) of Ringer solution was NaCl 111-2, KCl 1-88, CaCl2 1-08, NaHCO3 2-38 and NaH2PO^ 0-07.

FROG TASTE CELL RESPONSE

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RESULTS

Resting potential and input resistance of taste cells When a fungiform papilla was impaled with a micro-electrode, the microelectrode tip became suddenly negative by about 30 mV with respect to the indifferent electrode, and upon withdrawal of the micro-electrode the potential was restored to the original level. But sometimes the negativity decayed rapidly during the impalement. The results obtained on such cells were discarded in the present paper. The negative potential shift upon impalement occurred with two or three steps, as shown in Fig. 2. The mean magnitude of the initial notch, determined in twenty-five experiments, was - 15-9 + 5-1 mV (± S.D.), while that of the final potential value was - 42'8 + 3-7 mV. Such a potential profile as obtained on impalement of a papilla is consistent with the histological picture of the sensory l

XAJ

E a

~~~SS_

__

1 sec

Fig. 2. Records showing potential changes appearing when a microelectrode was advanced to impale a taste cell in a fungiform papilla. A, notch possibly originating from an associate cell in the sensory disk in the papilla; S, membrane potential from a sensory (taste) cell. In the right hand record the two S waves were probably due to successive penetration of two sensory cells. In this and subsequent records positivity at the microelectrode is displayed by upward deflexion. Sudden negative shift at the left and sudden positive shift at the right of each record indicate penetration of a cell with a microelectrode and its withdrawal from the impaled cell, respectively.

disk of a frog fungiform papilla, presented by DeHan & Graziadei (1971) and Graziadei & DeHan (1972). They demonstrated that the sensory disk was made up of two types of cells, the first cell type (associate cell) occupying the upper third portion of the disk and the second cell type (sensory cell) being located in the two lower thirds and extending its processes to the surface of the disk. Therefore, on advancement of a micro-electrode into the sensory disk, the electrode would first penetrate the associate cell, thus producing a notch at the falling phase of the negative shift, and then it would be inserted inside the cell body of a taste cell. Therefore, the final maximum potential value would represent the resting potential of a taste cell. Electrotonic potentials in response to injection of repetitive electrical

N. AKAIKE, A. NOMA AND M. SATO 92 pulses appeared immediately after impalement of the cell. The input resistance of the taste cell, which is mostly attributable to the resistance of the cell membrane and could be estimated from the magnitude of the electrotonic potential and current applied, varied widely from one cell to another. The mean resistance value (± S.D.) obtained from sixty-three cells, adapted to 0-01 M-NaCl and showing a mean ( ± S.D.) resting potential of -33-2 + 6-9 mV (range 25 to -46 mV), was 17-3 + 6-9 MD (range 6-46 MQ). Cell

I1 M sucrose

0.O m-H1-1

Cell2

0.02M quinine

0.1 m-NaC

0.02M quinine

01 m-NaCl

>

0001 -HCI 1 M sucrose (| E

INE

Fig. 3. Depolarizations produced by 041 M-NaCl, 0-02 M quinine, 0-001 MHC1 and 1 M sucrose in two different cells. In this and Fig. 6 the upper trace represents potential change in the cell recorded intracellularly, while the lower trace indicates currents applied into the cell through the microelectrode. Horizontal bars underneath or above the potential record show periods of application of stimuli.

The input resistance of a taste cell is reduced in magnitude with a spontaneous decrease in the resting potential level. The relation between the resting potential magnitude and the input resistance in a number of cells, in which the resting potential magnitude decayed spontaneously, is almost linear, and the regression equation of the resistance (y) on the resting potential (x) is represented by y = 04115x+5 165. Therefore, a decrease in the resting potential by 10 mV leads to a decrease in the resistance by about 13 % of the original value and a decrease of the former by 20 mV to a decrease of the latter by 17 %. Depolarizations produced by chemical stimuli in taste cells Almost all taste cells yielded sustained depolarizations in response to various chemical stimuli of sufficient concentrations. As shown in Fig. 3, NaCl, HCl and quinine produced marked depolarizations in a single taste cell, but response to sucrose is very small. The depolarization usually attained a peak with a rise time of a few seconds and maintained nearly a

93 FROG TASTE CELL RESPONSE constant value during stimulation, but those produced by quinine and HCl had a relatively long rise time. Little decrease in amplitude was observed during application of a stimulus. Therefore, the magnitude of response was indicated by the maximum magnitude or the steady-state magnitude. The rise time varied with change in the rate of flow of the solution. A quicker rate of flow yielded a faster rate of rise of the depolarization than that produced by the slower rate, although the final magnitudes of the depolarizations are similar to each other. The magnitude of the depolarizing response of taste cells to a chemical stimulus varied also with a change in stimulus concentration and quality. Increase in concentration resulted in an increase in the magnitude of response as well as the rate of rise of response.

Stimulus-response relations Relationships between the stimulus concentration and the magnitude of response to NaCl, KC1, CaCl2, HC1, quinine and sucrose, obtained from a number of cells, are shown in the upper half of Fig. 4. In this Figure the threshold concentration for NaCl is just above 0 01 M, because the tongue was adapted to 0-01 M-NaCl solution. KCl threshold appears to be also around 0-01 M. NaCl and KCl below 0-01 M produced a hyperpolarizing response. The thresholds for CaCl2, HC1, and quinine are very low, the order of 10-5 M. On the other hand, sucrose showed a very high threshold (025-0-5 M) and yielded a small response. In the lower half of Fig. 4 relationships between concentrations of stimuli applied to the tongue adapted to 0-01 M-NaCl and magnitudes of glossopharyngeal nerve responses are presented. Comparing the two Figures in Fig. 4 with each other it can be seen that the concentration-response relations for each stimulus are very approximately parallel to each other. The similarity between the two figures suggests a causal relationship between the depolarizations in taste cells and neural responses, and indicates that depolarizations in taste cells are the precursors for the initiation of impulses at the sensory nerve terminals.

Response profiles of taste cells to various kinds of stimuli Depolarizations produced by 003 M-NaCl, 0-01 M quinine, 0-001 MHCI and 05 M sucrose in fifty-one cells adapted to 0.01 M-NaCl are shown in Fig. 5. The resting potentials of these cells adapted to 0.01 M-NaCl had been initially - 35-7 + 5*7 mV on the average, but decreased to - 28-8 + 5-8 mV just before chemical stimulation of cells. The resting potential values in individual cells measured just before the stimulation are shown at the bottom of Fig. 5. As shown in this Figure, marked responses were obtained to NaCl, quinine and HCl in nearly all the cells.

N. AKAIKE, A. NOMA AND M. SATO 94 However, response to sucrose was very poor. The average magnitudes (+ S.D.) of responses to NaCl, quinine and HCl in Fig. 5 are 9-9 + 2-5, 8 3 + 3 2 and 17O0 + Ib5 mV, respectively. The magnitudes of responses to these three stimuli varied from one cell to another, and the variation is marked in the HCl response, but small in the one to NaCl. As shown in this Figure, the response profile for one stimulus varied from that for another. When correlation coefficients between responses to pairs of 25 E 20 c 15

o

NaCI (11)

*KCI (9)

^A HCI (11) CaC12 (9)

LA Quinine (10)

10 5

Sucrose (5)

0.5 of

5'

C

EF

00o NaCI (9) KCI (9) DO AO0 HC1I(3) A

31DO

E 2'00 z

| o

-

Quinine (4) 0 CaCI2 (9) Sucrose

(5)

1' 01 10-i 10-6

10-3 10-2 10-1 Concentration (M) Fig. 4. Relationships between taste stimulus concentration and response magnitude, obtained from the taste cell (upper graph) and from the glosso. pharyngeal nerve (lower graph). Stimuli were indicated by different symbols. Each point in the Figure represents the mean value of the maximum magnitude of receptor potentials obtained from several experiments, the number of which is indicated by a numeral inside a parenthesis after each stimulus. In all the experiments taste receptors were preadapted to 0.01 MNaCl before each stimulation.

10-4

stimuli across fifty-one cells were calculated, a significant negative correlation was found only between responses to NaCl and HCl (rNaCl-HcI = -0-36, 0001 < P < 0-01, t test), but correlations between other pairs are not significant (rNaCI-quinine = 0,32, rquinine-HM = 0.06). Also correlations between resting potentials and responses to either one of the stimuli is low: rNacl-r.p. = -016 (P < 05), rquinine-r.p. = 0-36 (0 01 < P < 0.05) and rHci-r p = 0 40 (0.001 < P < 0 01). In the above experiments winter frogs were used as materials. Since

95 FROG TASTE CELL RESPONSE it has been reported that the glossopharyngeal nerve response to sucrose is smaller in magnitude in winter frogs than in summer frogs (Yamashita, 1964), responses to 1 M sucrose and 0 1 M-NaCl were recorded from thirty 1

0003 m-NaCl

10

1Fo

0 20

0-01

M-quinine

1A0

>-E

hi

C

20

0.

0.

0001Ms-HCIro

0

I0 U

Ce

3

I0

to 1 I0

0' 2 1m

m sucrose

rI

40-

E

10 C

0

_,

._

0~ be -' to

C

-

l0

._

0

10

20

30

40

50

Taste cells

Fig. 5. Responses to the four basic stimuli and resting potentials in fiftyone taste cells, which were adapted to 0-01 M-NaCl. Experiments were carried out with hibernating frogs in winter.

taste cells in summer frogs. The results indicated that 70 % of all the cells in summer frogs responded to 1 M sucrose, although the magnitude of depolarizations was still only a few millivolts. Taste cells yielded good responses to a variety of salts. Response of fifty-two cells to 0-03 M-NaCl, 0-03 m-KCl, 0 03 M-MgC12 and 0-01 m-CaC12 4

PH Y

254

96

N. AKAIKE, A. NOMA AND M. SATO were compared with one another. In general 0-01 M-MgCl2 and CaCJ2 produced responses greater than those to 0 03 M-NaCl and KCl, and responses to the former two were similar in magnitude to each other. Among the four salts KCI was least effective. The mean magnitudes (± S.D.) of responses to NaCl, KC1, MgCl2 and CaCl2 are 9-4 + 2-8, 6-8 + 2.1, 15-0 + 5-6, and 15-5 + 5.9 mV, respectively. The response profile for CaCl2 was very similar to that for MgCl2. When correlation coefficients between responses to pairs of these four stimuli across fifty-two cells were calculated, they were found to be high (rNacl-Kcl 0,75, rNaCl-caCl2 = 0'60 rNac1~Igc12 = 0*74, r KCL CaC12 = 0*57, rKC1- MgC12 = 0-60, rcac 0Mgc1 = °88). However, correlation between resting potentials and responses to either one of the four stimuli was low and not significant as judged by t test. Responses of twenty cells to 1 mM-HCl, acetic acid (A.A.), tartaric acid (T.A.) and 0-1 M-NaCl were also examined, and all the cells were found to yield responses to the three acids. Among the acids HCl was most effective while acetic acid was least effective: the mean magnitudes (± S.D.) of depolarizations produced by HCl, acetic acid, tartaric acid and NaCl were 16-9 + 7-2) 8-5 + 4 9, 14-4 + 5-2 and 13-8 + 541 mV, respectively. The response profiles of twenty cells for acids are similar to one another, but differ from that for NaCl. Correlation coefficients between amounts of responses to pairs of acids across cells are very high (rHICL-A.A = 0 88, rHC-T.A. = 0,88, and rA.A.-T.AA = 0.81), but the coefficients between NaCl and either one of the acids are very low (rNaclHcl = 0 16, rNac1AA = 0-10, and rNaCl T.A = -0.22).

Responses of taste cells adapted to Ringer solution The magnitude of resting potential in the taste cell varies with variation in NaCl concentration in the adapting solution. As described above, when taste cells were adapted to 0-01 M-NaCl solution, the magnitude of resting potential is about -35 mV on the average. On the other hand, taste cells adapted to Ringer solution had a resting potential of - 24-4 + 545 mV (mean + S.D. of twenty-three cells). This is mainly attributed to a depolarizing effect by a high NaCl concentration in Ringer solution. Also the magnitudes of responses to taste stimuli are smaller in Ringer solution than in 0-01 M-NaCl, and cells adapted to Ringer solution showed a more pronounced differential sensitivity to the stimuli. Although many cells responded to all the stimuli, there were a few cells that responded to either KCl alone, or MgCl2 and CaCl2 only. Therefore, correlation coefficients between responses to pairs of salt stimuli across taste cells adapted to Ringer solution are poor (rNaci-Kci = 055, rNaci-caC12 = 0-48, rKCi-CaC12 = 0-14, rKCI-MgCI2 = 0.01, rCaMC2 = 0 85) compared with those obtained from cells adapted to 0-01 M-NaCl.

FROG TASTE CELL RESPONSE

97

Response to water It has often been reported that application of tap water or pure water on the frog tongue elicited a massive volley of impulses in the glossopharyngeal nerve (Andersson & Zotterman, 1950; Koketsu, 1951; Kusano & Sato, 1957; Nomura & Sakada, 1965). In the present study responses of taste cells to tap water, deionized water, 1 mM-NaCl and 6 mM-CaCl2 0-01 M-Na Cl 0006 M-CaCl2

De-ionized 0-001 m-NaCI Tap water water

Ringer

0-001 m-NaCI Tap water De-ionized water

0-006 M-CaC12

10 sec

Fig. 6. Responses to 1 mM-NaCl, 6 mM-CaCl2, tap water and deionized water in two taste cells, one being preadapted to 0 01 M-NaCl and the other to Ringer solution.

were examined, when they had been adapted to 0.01 M-NaCl and Ringer solution. Potential changes produced by these stimuli in taste cells adapted to both kinds of solutions are shown in Fig. 6. As shown in this Figure, both tap and deionized water produced hyperpolarizations in both kinds of cells as did 1 mM-NaCl, while 6 mM-CaCl2 yielded a small depolarization. The hyperpolarization produced by 1 mM-NaCl and tap water is greater in the cell adapted to Ringer solution than in the one adapted to 0'01 MNaCl, while the hyperpolarization produced by deionized water is greater in cells adapted to 0-01 M-NaCl. In the latter cells the hyperpolarization decreased in magnitude immediately after application of deionized water. No depolarizing response to water was demonstrated in the present 4-2

N. AKAIKE, A. NOMA AND M. SATO study, although recordings of response to water were made from cells inside fungiform papillae over various parts of the surface of the tongue. 98

Conductance change during chemical stimulation As can be seen in Fig. 3 (cell 2), the electrotonic potential superposed on the depolarizing response to NaCl is small in magnitude compared with that during the resting state. On the other hand, such a prominent reduction in the magnitude of electrotonio potentials could not be seen during stimulation by quinine or HCl in Fig. 3. The reduction in the electrotonic potential magnitude was found to become more marked when a higher concentration of NaCl was employed. Since responses to various kinds of stimuli of varying concentrations in taste cells were recorded in the experiments as shown in Fig. 4 (top part of Figure), relationships between the magnitude of depolarizations produced by stimuli of different concentrations and the relative conductance magnitude (the ratio of the electrotonic potential magnitudes between the rested and stimulated state), obtained during chemical stimulation of taste cells, are shown in Fig. 7. It is clearly shown in this Figure that NaCl and KCl produced a marked increase in the relative conductance accompanying the depolarizing response. On the other hand, a decrease in the conductance is associated with a hyperpolarization, produced by 3 mm-NaCl and KCl. The relative increase of the conductance during stimulation by CaCl2 is much smaller than that produced by NaCl and KCl, although the depolarization produced by the former stimuli is nearly the same as that yielded by the latter (Fig. 7). As shown in Fig. 7, virtually little increase in the conductance was produced by quinine and HCl, which yielded depolarizations as large as 15 mV. Also 1 mm acetic and tartaric acids produced little change in the relative magnitude of conductance, although they yielded, on the average, depolarizations of 8'5 and 14-4 mV, respectively. Scarcely any change in the relative conductance magnitude was produced by 1 M sucrose, which yielded a few millivolts depolarization.

Current-voltage relationships In order to examine more precisely how the membrane conductance of a taste cell varies by application of various chemical stimuli, currentvoltage relationships were determined in four taste cells adapted to different solutions. Examples of the I-V relations are demonstrated in Fig. 8, where all the relations are linear, indicating that the taste cell membrane behaves as an ohmic resistance. As shown in Fig. 8, the slope of the straight line is smaller for 0-1 M-NaCl than for 0 01 M-NaCl, indicating a decrease in the input resistance of the cell from 17 to 11 MD, while it is larger for 0-001 M-NaCl than for 0.01 M-NaCl, the input resistance

999 FROG TASTE CELL RESPONSE being increased from 17 to 26 MQ. Similarly, the input resistance is decreased from 30 to 22 MU and from 27 to 21 MU by changing the adapting solution from 0-01 M-NaCl to 0-03 M-CaCl2 and 0-001 M-HCl, respectively. On the other hand, the input resistance is increased from 17 to 33 MQ by changing the solution from 0T01 M-NaCl to 0-02 M quinine. 2-0

o

a_ A*HCI (1 1)/ *Quinine (10) O~1 CaC12 (1 5)_

X 1-6

-5

NaCI (11)

0

5 10 Receptor potential (mV)

is

20

Fig. 7. Relationships between the receptor potential amplitude and the relative conductance magnitude. The latter represents the ratio of the electrotonic potential magnitudes between rested and stimulated state. The data in this Figure were taken from the experimental results shown in the upper portion of Fig. 4.

The results of series of the experiments to determine the input resistance of taste cells in varying adapting solutions by measuring the I- V relations are shown in Table 1. As shown in this Table, increase in NaCl concentration from 0-01 to 0-1 M resulted, on the average, in a depolarization of 20 mV and a decrease in the input resistance or an increase in the conductance, while a hyperpolarization as well as a decrease in the conductance occurred by reducing NaCl concentration. Changing the adapting solution from 0-01 m-NaCl to 0 03 M-CaCl2 or 0 001 M-HCl brought about also a depolarization of about 20 mV and an increase in the conductance, but the magnitude of increase in the conductance is small compared with that associated with the depolarization by 0-1 M-NaCl. On the other hand, 0 01 M quinine produced a decrease in the conductance by about 25% associated with a depolarization of 18 mV.

Variation in input resistance with change in membrane potential To see how change in the membrane potential affects the input resistance or the relative conductance of a taste cell, variation in the input

N. AKAIKE, A. NOMA AND M. SATO resistance due to change in the membrane potential magnitude was investigated on four taste cells by injecting repetitive short pulses superposed on a very long current pulse of either polarity but of varying intensity. The results indicate that the relative conductance is increased, on the average, by 12% with 10 mV depolarization, and by 26% with 20 mV depolarization, while it is decreased by 11 % with 10 mV hyperpolarization. The results are in approximate agreement with those obtained on spontaneously depolarizing cells: 100

-60 mV

-10 I ( x 10-10 A) -20

*-30

-40

2

Fig. 8. Current-voltage relationships obtained from four taste cells adapted to varying solutions. Adapting solutions are indicated in each Figure. In all the Figures, magnitudes of electrotonic potentials (mV) are plotted as ordinates against currents applied (x 10-10A) as abscissae. In each Figure the resting potential magnitude and the input resistance, calculated from the slope of each straight line, are indicated by mV and MO, respectively.

FROG TASTE CELL RESPONSE

101

TABLE 1. Changes in the resting potential and the input resistance of taste cells with change in the adapting solution

Resting potential (mV) -33-2 + 6-4 -13-6 5-8

Input resistance (MO) 14-6 + 3-8 8-3 + 2-5 21-6+5-6 23-4 + 5-3 17-8 + 4-4 18-9 6-6 14-9 +48 18-6 ±70 24-6 +5-3

Relative Solution conductance 0.01 M-NaCl 1.00 0 1 M-NaCl 1-76 0001 M-NaCl -51-0±4-8 0-68 0 01 M-NaCl -31-7 +57 1 00 0 03 M-CaCl2 -12-8 + 5-4 1-31 0.01 M-NaCl -28-2 + 2-5 1 00 0 001 M-HCl -10-7 +5-7 1-27 0 01 M-NaCl -31-3 +32 1 00 0-02 M quinine -13-5 5-1 0-76 Each value indicates the mean + S.D. of determinations of six cells.

Change in receptor potential magnitude with variation in membrane potential The receptor potentials of a taste cell produced by 0.1 M-NaCl, 0-03 mCaC12, 0-02 M quinine hydrochloride and 0-001 m-HCl at varying levels of the membrane potential were recorded. Examples of the relationships between the membrane potential magnitude and the receptor potential amplitude for the four kinds of stimuli are shown in Fig. 9. The amplitude of the receptor potential produced by 0.1 M-NaCl decreased linearly with a rise in the membrane potential, and became zero at about +20 and + 40 mV in the Figure, reversing its polarity above these levels. The mean value of the reversal point obtained from the four experiments is + 26 mV. The amplitude of the receptor potential produced by 0 03 M-CaCl2 is also decreased linearly with a rise in the membrane potential, and reverses its polarity at the membrane potential of + 10 and + 20 mV in Fig. 9. The mean value of the reversal point obtained from the four experiments is +7 mV. The relationships between the membrane potential level and the receptor potential amplitude for 0-001 M-HCl and 0-02 M quinine are quite different from those for NaCl and CaC12. As shown in Fig. 9, the amplitude of the receptor potential produced by HC1 is scarcely changed with variation in the membrane potential from + 40 to -60 mV. In two cells among the four examined the receptor potential amplitude did not vary, although the membrane potential was lowered to -80 mV. In two other cells the receptor potential amplitude declined with a fall in the membrane potential level below -60 mV, and reversed its polarity at about -60 and

N. AKAIKE, A. NOMA AND M. SATO 102 -85 mV. The change in the receptor potential amplitude in one of the two cells is demonstrated in Fig. 9 by triangles. The maximum amplitude of the receptor potential generated by quinine is affected relatively little by variation in the membrane potential, but 0-03 M-CaC12

I 40 30

20 10 -100

-50

-50

-100

0

0.001 M-HCI

0.02 M quinine

-50

* 20

* A.

10

A_ -100

0 -10

--10

(mV)

- 10 a -100 Id so

A

^

l

|

|

-10

0 -10

-20

-20

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-so

so

-30 Fig. 9. Relationships between the amplitude of receptor potentials -30

(ordinates), produced by 0-1 M-NaCl, 0-03 M-CaCl2, 0-02 M quinine and 0-001 M-HCl, and the steady membrane potential abscissaee). Triangles and circles in each Figure represent two different cells, and filled triangles and circles show the amplitude of the receptor potential in cells to which no current was applied. A

B 71

D

C 1-

7I

10 sec Fig. 10. Receptor potentials in a taste cell, elicited by 0.02 M quinine at four different membrane potential levels. The upper trace indicates current applied to the cell to modify the membrane potential, while the lower trace shows the transmembrane potential recorded from the cell. Horizontal bar underneath each record indicates the period of application of stimuli.

103 FROG TASTE CELL RESPONSE there is an indication that the former decreases slightly with an increasing hyperpolarization (Fig. 9). This is because of a decline in the amplitude of the receptor potential with time during quinine action on hyperpolarized cells. As shown in Fig. 10, the receptor potential declines rapidly immediately after attaining a peak (Fig. 10 C) when the cell had been hyperpolarized by about 50 mV, and the decline is more marked with a greater hyperpolarization (Fig. lOD). DISCUSSION

Resting potential and responses to chemical stimuli in taste cells The present study indicated that the taste cell has a resting potential of about -35 to -45 mV on the average, when measured in 0*01 M-NaCl, but it was about -25 mV in Ringer solution. Sato (1969, 1972) reported the mean values of resting potentials of taste cells in the dissected tongue of R. nigromaculata and R. catesbeiana, which had been adapted to Ringer solution, to be -21 mV. This is similar to the value obtained under similar conditions in the present study. Sato (1969, 1972) reported that frog taste cells responded well to 0-5 m-NaCl, 4 mM quinine, 16 mM acetic acid and 0-25 M sucrose, and his paper (1972) indicated that eight cells among eleven yielded a response to 0-25 M sucrose. Although our study demonstrated very good responses in taste cells to NaCl, quinine, acids and various other salts, responses to sucrose were poor. In earlier studies responses in the glossopharyngeal nerve of the frog to sucrose were examined by a number of investigators, but they were reported to be very small (Kusano & Sato, 1957), especially in hibernating frogs (Kusano, 1960) and at low temperature (Yamashita, 1964). Therefore, our study indicating a poor response to sucrose in taste cells especially in winter frogs is in agreement with the results of the earlier studies on the glossopharyngeal nerve response to sucrose. The unusually high sensitivity of the frog gustatory receptors to calcium salts has been noted by a number of investigators (Casella & Rapuzzi, 1957; Kusano, 1960; Yamashita, 1963, 1964; Nomura & Sakada, 1965). The threshold concentration of CaCl2 for eliciting impulses in the glossopharyngeal nerve when applied to the tongue has been reported to be about 01 mM (Casella & Rapuzzi, 1957; Yamashita, 1964). Such a low threshold concentration of CaCl2 for eliciting responses in the glossopharyngeal nerve and in taste cells was also observed in the present

study.

104

N. AKAIKE, A. NOMA AND M. SATO

Mechanisms underlying the membrane potential change in taste cells The results of the present experiments have demonstrated that frog taste cells behave as a purely ohmic resistance in response to injected currents and that not only the membrane potential but also the input resistance in a cell vary in magnitude with variation in the stimulating or adapting solution. Both a depolarization and a decrease in the resistance occur with NaCl, KCl, CaCI2 and HCl above a critical concentration, while a hyperpolarization and an increase in the resistance are produced by NaCl below that concentration. Quinine yielded a unique effect in that it produced an increase in the resistance associated with a depolarization. These observations are essentially similar to those reported by Ozeki (1971) on taste cells in fungiform papillae of the rat, indicating that basic properties of the taste cell membrane are the same for the frog and rat. The decrease in the input resistance is marked during depolarizations by NaCl and KCl compared with that produced by CaCl2 and acids. Since a decrease in the input resistance in taste cells indicates an increased membrane permeability to ions during application of a chemical to the cell, ionic permeability of the membrane would be involved in the generation of the cell membrane potential change by NaCl and KCl. The linear relationship between the amplitude of the receptor potential produced by NaCl and the steady membrane potential level in taste cells is analogous to the one between the end-plate potential and the membrane potential (Fatt & Katz, 1951), and supports further the proposition that the receptor potential in response to NaCl is generated as an increase in the membrane permeability to ions, possibly Na and chloride. Although CaCl2 is more effective in producing a depolarization than are NaCl and KCl, the former produces a much smaller amount of resistance change than that generated by the latter salts with a similar amount of depolarizations. Such a fact suggests that CaCI2 exerts an action on the taste cell membrane qualitatively different from those produced by NaCi and KCl. Since the amount of resistance change produced by CaCl2 is nearly equal to that found during spontaneous and current-induced depolarizations of the cell of a similar magnitude, there is a possibility that the resistance change brought about by CaC12 would result mostly from a depolarization of the membrane, which is generated by the action of CaCl2 on the taste cell membrane such as adsorption of Ca ions on the membrane structure. The linear relationship between the amplitude of the receptor potential produced by CaCl2 and the membrane potential suggests that interaction between calcium ions and receptor sites in the membrane is dependent on the potential difference across the membrane.

105 FROG TASTE CELL RESPONSE The action of HCl and other acids on taste cells is similar to that of CaCl2 in that both produce a relatively small decrease in the input resistance, associated with a depolarization. Therefore, the decrease in the input resistance may be a consequence of the depolarization, which is produced by interaction of hydrogen ions with the membrane receptor sites. However, the depolarization produced by a given concentration of HCl is scarcely influenced by variation in the membrane potential level over a wide range. This differs from the situation for CaCl2, and indicates that the mechanism of interaction between hydrogen ions and receptor sites are essentially different from that occurring between Ca ions and receptor sites. Quinine depolarizes a cell with an increase in the input resistance. As suggested by Ozeki (1971), one explanation may be that quinine reduces the membrane permeability to potassium ions in the taste cell as in the muscle fibre (Falk, 1961), thereby producing the depolarization. The effect of quinine on the taste cell is also different from those of NaCl and CaCl2 in that the magnitude of the receptor potential produced by quinine is influenced relatively little by variation in the membrane potential level over a wide potential range, as observed by Ozeki (1970, 1971) on the rat taste cell. However, the depolarization produced by quinine in the frog taste cell hyperpolarized by about 50 mV decays relatively rapidly after attaining the maximum (see Fig. 10). It suggests a change in the properties of the receptor mechanism that reacts to quinine, but little satisfactory explanation regarding its nature can be given at the moment.

Response to water Application of tap water or pure water on the frog tongue elicits a massive volley of impulses in the glossopharyngeal nerve (Andersson & Zotterman, 1950; Koketsu, 1951; Kusano & Sato, 1957; Nomura & Sakada, 1965). Nomura & Sakada (1965) assume that the response to tap water is caused by Ca ions in the water, because of a very low threshold of frog taste receptors for CaCl2 and of the similarity of the magnitude of response to CaCl2 and that for tap water having a similar Ca concentration. However, in the present experiments CaCl2 yielded a depolarization in the cell, while tap water and deionized water produced a hyperpolarization as did 1 mM-NaCl. Therefore, the effect of water on taste cells is entirely different from that of weak CaCl2 solution, although the magnitude of hyperpolarization produced by tap water is smaller than that by deionized water and this may be attributed to some ions dissolved in tap water. Sato & Beidler (1973) reported the presence of two types of cells in the fungiform papilla of the frog tongue, one which shows a depolarization in response to concentrated NaCl solution and a hyperpolarization to

106 N. AKAIKE, A. NOMA AND M. SATO water (NS-cell)while the other one that responds to water with a depolarization and to concentrated NaCl solution with a hyperpolarization (WS-cell). However, in the present experiments no latter type of cells was found, but all the cells examined yielded a hyperpolarization in response to water and a depolarization to concentrated NaCl solution. At the moment, neither the discrepancy between the results of the two sets of experiments can be explained satisfactorily, nor the question how the hyperpolarization in the cell produced by application of water does initiate impulses at the afferent nerve terminal can be answered. These problems should be answered by further experiments.

Discrimination of stimulus quality at taste cells Sensitivities to varying kinds of chemicals have been demonstrated in a single taste cell of the frog (Sato, 1972) and of the rat (Kimura & Beidler, 1961; Ozeki & Sato, 1972). The present study has also indicated that almost all taste cells possess good sensitivities to salts, acids and quinine when they have been adapted to 0.01 m-NaCl, and that differential sensitivity to the chemicals is higher in cells adapted to Ringer solution than in those adapted to 0.01 m-NaCl. The present study has further demonstrated that the response profiles across cells for two similar kinds of stimuli such as CaCl2 and MgCl2 are similar to each other, indicating a high correlation coefficient between the amounts of responses to pairs of stimuli, while different kinds of substances such as NaCl and HCl yield the response profiles different from each other or a low correlation coefficient between responses to the two stimuli. These results indicate that discrimination of stimulus quality at the taste cells may be made by difference in the response pattern across cells. This work was supported by grants from the Ministry of Education in Japan (grant nos. 811009 and 911109). REFERENCES

AKAIKE, N., NOMA, A. & SATO, M. (1973). Frog taste cell response to chemical stimuli. Proc. Jap. Acad. 49, 464-469. ANDERSSON, B. & ZOTTERMAN, Y. (1950). The water taste in the frog. Acta physiol. scand. 20, 95-100. BEIDLER, L. M. (1953). Properties of chemoreceptors of tongue of rat. J. Neurophysiol. 16, 595-607. CASELLA, C. & RAPuZZI, G. (1957). Azione dell'acqua del CaCl2 e del NaCl sui recettori linguali nella rana. Arch. Sci. biol. Bologna 41, 191-203. DEHAN, R. S. & GRAZIADEI, P. P. C. (1971). Functional anatomy of frog's taste organs. Experientia 27, 823-826. FALK, G. (1961). Electrical activity of skeletal muscle. In Biophysics of Physiological and Pharmacological Actions, ed. SHANES, A. M., pp. 259-279. Washington, D.C.: Am. Assn. for the Advancement of Science.

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FATT, P. & KATZ, B. (1951). An analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115, 320-370. FRANK, K. & BECKER, M. C. (1964). Microelectrodes for recording and stimulation. In Physical Techniques in Biological Research, vol. 5, ed. NASTUK, W. L., pp. 22-87. New York: Academic Press. GRAZIADEI, P. P. C. & DEHAN, R. S. (1972). The ultrastructure of frog's taste organs. Acta anat. 80, 563-603. KIMURA, K. & BEIDLER, L. M. (1961). Microelectrode study of taste receptors of rat and hamster. J. cell. comp. Physiol. 58, 131-140. KOKETSU, K. (1951). Impulses from receptors in the tongue of a frog. Kyushu Mem. med. Sci. 2, 53-61. KUSANO, K. (1960). Analysis of the single unit activity of gustatory receptors in the frog tongue. Jap. J. Physiol. 10, 620-633. KUSANO, K. & SATO, M. (1957). Properties of fungiform papillae in frog's tongue. Jap. J. Physiol. 7, 324-338. NOMURA, H. & SAKADA, S. (1965). On the 'water response' of frog's tongue. Jap. J. Physiol. 15, 433-443. OZEKI, M. (1970). Hetero-electrogenesis of the gustatory cell membrane in rat. Nature, Lond. 228, 868-869. OZEKI, M. (1971). Conductance change associated with receptor potentials of gustatory cells in rat. J. gen. Physiol. 58, 688-699. OZEKI, M. & SATO, M. (1972). Responses of gustatory cells in the tongue of rat to stimuli representing four taste qualities. Comp. Biochem. Physiol. 41A, 391-407. SATO, T. (1969). The response of frog taste cells (Rana nigromaculata and Rana catesbeiana). Experientia 25, 709-710. SATO, T. (1972). Multiple sensitivity of single taste cells of the frog tongue to four basic taste stimuli. J. cell comp. Physiol. 80, 207-218. SATO, T. & BEIDLER, L. M. (1973). Relation between receptor potential and resistance change in the frog taste cells. Brain Res. 53, 455-457. YAMASHITA, S. (1963). Stimulating effectiveness of cations and anions on chemoreceptors in the frog tongue. Jap. J. Physiol. 13, 54-63. YAMASHITA, S. ( 1964). Chemoreceptor response in frog, as modified by temperature change. Jap. J. Physiol. 14, 488-504.