Characteristics of the transepithelial potential change produced by NACl in bullfrog tongue

Camp. Biochem. Physiol. Vol. 82A, No. 2, pp. 361-366, 1985 Printed in Great Britain

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CHARACTERISTICS OF THE TRANSEPITHELIAL POTENTIAL CHANGE PRODUCED BY NaCl IN BULLFROG TONGUE HIROYUKISORDAand Funo SAKUDO Department of Physiology, Fukuoka Dental College, Sawara-ku, Fukuoka, Japan (Received 22 January 1985)

Abstract-Isolated dorsal epithelium from a bullfrog tongue exhibited changes in potential difference across the tissue when NaCl was applied to the mucosa after adaptation to Ringer’s solution. 2. Characteristics of the potential change were similar in many respects to those in single taste cells of frogs. 3. The conclusion is that the potential change may influence taste sensation.

INTRODUCTION The application of NaCl or other chemicals to the tongue produces a slow, depolarizing potential within taste cells (receptor potential) in the tongues of rodents (Kimura and Beidler, 1961; Ozeki and Sato, 1972) and of amphibians (Sato, 1969; Sato and Beidler, 1975; Eyzaguirre et al., 1972; Akaike et al., 1976; West and Bernard, 1978). The receptor potential characteristics of various agents and their concentrations are very similar in appearance to taste nerve activity (Kimura and Beidler, 1961; Akaike et al., 1976). Taste reception is considered generally to originate only from the receptor potential within the taste cells of taste organs which are scattered mainly on the dorsal surface of the tongue. The morphology of the taste organs is distinctly different from the surrounding ciliated epithelium. The taste cells of the organs are also unique because they contact both the dorsal surface and the taste fibres (Murray, 1971). The ciliated epithelium, occupying most of the total area of the dorsal surface, ignores chemicals, being an effective barrier to them (Beidler, 1967; Mistretta, 1971; Kamo et al., 1974). However, as was suggested by Taglietti et al. (1971), Hayashi (1978) first reported that gustation in frog tongue was significantly influenced by potassium penetration through the epithelium. Furthermore, DeSimone et al. (1981, 1984) demonstrated that the dorsal epithelium of the dog tongue showed a rapid increase of ion penetration, measured by either an open-circuit potential or short-circuit current, when a hypertonic NaCl solution was placed on the mucosal surface. This suggests a possible link to gustatory transduction. We reported in a previous paper (Soeda and Sakudo, 1985), that stimulation of bullfrog tongue with NaCl or other chemicals produces a slow change in transepithelial potential difference across the dorsal epithelium, which resembles the receptor potential in single taste cells. In the present study, we not only demonstrate that the change in potential difference induced by NaCl is in many respects similar to that

of the receptor potential, but also that it is produced by passive ion transport through the tissue. MATRRULSAND METHODS Preparations All experiments were carried out using bullfrogs (Rana catesbeiana), weighing 200-350 g. The tongue was isolated from the frogs which had been decrebratcd and pithed. The tongue was washed with Ringer’s solution to remove the mucus sutliciently and then pinned, dorsal surface down, to a soft plastic board. The frenulum and ventral epithelium were isolated from the underlying tissue with forceps and scissors. To obtain the dorsal epithelium, all the muscle fibrcs were removed from the rostra1 to caudal sides, along the direction of the fibres, but cutting through the connective tissue between the muscle and the epithehum. These procedures were undertaken with particular care under a binocular microscope to avoid injuring the dorsal epithelium. The fine structure of the dorsal epithelium was examined under light microscopy. Figure 1 shows the section of the epithelium chosen to reveal a single taste organ and its surrounding ciliated epithelium. Comparing this photograph with one of rat epithelium (Mistretta, 1971), it has no keratinixed layer covering the surface, particularly the ciliated epithelium. Toyoshima et al. (1984) found the same thing in their study of a single bullfrog taste organ. Recording apparatus The potential differences across the tissue were measured using a modified Ussing chamber (Ussing and Zerahn, 1951), as shown in Fig. 2. The dorsal epithelium was mounted between two plastic boards, each with a frame around a 1 cm* elliptical-shaped hole, so as to isolate the mucosal and serosal surfaces. Sufficient pressure was applied to tbe boards with small clips in order to achieve a leak-proof seal. Both surfaces of the tissue were continuously perfused with aerated Ringer’s solution at the rate of 3 ml per min. Comparisons were made between the dorsal epithelium and other epithelia by the use of same apparatus. To record the potential difference, agar-1 M KC1 electrodes, with an Ag-AgCl wire in 1 M KC1 solution, were placed immediately below each surface. One of the electrodes on the serosal surface was grounded. The electrodes were connected to a high impedance- and DC-amplifier and a pen recorder.

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Fig. 1. Photograph of a cross-section of the dorsal epithelium in bullfrog tongue. Magnification: x 230. A single taste organ composed of taste, and other, cells is seen at right in the middle (arrowhead). The organ is surrounded by ciliated epithelium in this section.

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Tissue resistance was measured with a Wheatstone bridge circuit employed between the mucosal and the serosal surfaces of the dorsal e&helium, a 1OOmsec pulse current was applied at the rate of 0.2-0.5 Hz through the tissue. The resistance could be estimated from the magnitude of the electrotonic potentials across the tissue and from the pulse currents applied. The bridge circuit was also used to change the resting potential difference between both surfaces, by applying a steady current through the tissue. Shunting resistance of the bridge was 100 KG, because the potential difference was unchanged, regardless of whether or not the bridge circuit was put in parallel with the input. Test solutions Test stimuli used were mainly NaCl. NaCl was admixed in Ringer’s solution for desired concentrations. other chemicals and drugs were also admixed in Ringer’s solution. For stimulation, the test solution was applied to the muwsal surface for 50 sec. The continuously irrigating Ringer’s solution and each test solution could be controlled by stopcocks beneath the irrigators as shown in Fig. 2. The composition of Ringer’s solution was NaCI 112.0 mM, KC1 2.0mM, CaCl, 1.8 mM, and NaHCO, 2.0 mM per liter water. RESULTS Resting potential

Fig. 2. Schematic representation of the experimental set-up. A: General scheme. B: Lateral view of the isolating apparatus and the mucosa or serosa of the dorsal bullfrog tongue epithelium: r.s., Ringer’s solution; t.s., test solution; d.e., dorsal epithelium (mucosa on right); el., agar-1 M KC1 electrode.

daxerence across dorsal epithelium

When Ringer’s solution only was applied to both surfaces of the dorsal epithelium, there was a small potential difference across the tissue indicated by a level shunted to the amplifier input. The potential difference varied from -4.3 to +2.5 mV on the mucosa to the serosa grounded, from one tissue to the

NaCl response across tongue epithelium

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Fig. 3. Records showing a change in transepithelial

response associated with a decrease in resistance between the mucosa and the serosa of the dorsal epithelium. Changes appear when 0.02-0.5 M NaCl were applied to the mucosa during the period represented by a horizontal bar in each record. Numbers to the left of each record show the NaCl concentration for stimuli. Upward direction of the responses indicates that the mucosa is electrically negative to the serosa grounded. The bottom record shows the pulse currents through the tissue.

other. The mean value obtained from 32 tissues was - 1.3 + 2.1 mV. This value is negligible in comparison with those of bullfrog skin (-76.3 + 18.9 mV) and the urinary bladder membrane (- 84.1 f 17.5 mV), which were measured by the same apparatus. The large potential difference across the skin coincides with the open-circuit potential measured by an Ussing chamber (Ussing and Zerahn, 1951). Changes in potential d@rence produced by NaCl stimulation

and tissue resistance

After a sufficient adaptation to Ringer’s solution, stimulating the mucosa with NaCl produced a slow change in the potential difference across the tissue. The mucosa became negative to the grounded serosa (NaCl response), associated with a decrease in tissue resistance. Both changes in the response and the resistance were restored upon withdrawal of the stimulant within l-5 min as shown in Fig. 3. The maximum magnitude of the NaCl responses was clearly dependent upon the stimulus concentration. With concentration below 0.2 M NaCl, the rising phase of the responses became steeper, but did not acheive a steady state. Increasing the concentration of NaCl up to 0.5 M resulted in a more rapid rising phase, followed by a steady state or an overshoot and a decline. The resistance inherent in the tissue itself varied from one tissue to another. The mean resistance obtained from 16 tissues was 710 + 180 Q/cm2 in the resting state. This value is similar to that obtained for dog tongue (DeSimone et al., 1981, 1984). The maximum decrease in the resistance was dependent upon both the stimulus concentration and the response magnitude. Although the maximum response magnitude and the decrease in the resistance varied with a change in

0.05 0.1 0.5 1 NaCl concentration (H) Fig. 4. Relationships of both the maximum changes in the response magnitude and in the tissue resistance to stimulus NaCl concentration in the dorsal epithehum. Open and solid circles indicate the response magnitude (Rm) and the relative values in tissue resistance (Tr), respectively. Each point and its vertical bar represent the mean values and their standard deviations derived from 16 tissues.

the flow of the test solution, there were similar responses with a rate of 3 ml per min or more. There was no response in the serosa when any concentration of NaCl was applied. Equimolar stimulation with salts similar to NaCl (i.e. KCl, CaCl?, NaNO, or Na,SO,) also produced responses assocrated with a decrease in resistance, similar to those noted with NaCl stimulation. Stimulants used in concentrations of 0.2 M each could be ordered according to the maximum response which each induced: CaCI,, KCl, NaCl, Na$O, and NaNO,. The NaCl response in the dorsal epithelium differed from the responses of other epithelia. Epithelia in ventral surface of the tongue and in the palate, which are exposed to oral cavity fluid just as is the dorsal epithelium in viuo, produced a small degenerative response with mucosal stimulation. The skin and urinary bladder membrane, having a large, negative resting potential difference, produced a large response in the positive direction. Stimulus changes lationships

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Relationships between NaCl concentration and the maximum changes in response magnitude and in tissue resistance, obtained from simultaneous recordings, as in Fig. 3, are shown in Fig. 4. Comparing the two sigmoidal curves in the figure, it can be seen that both concentration-response and resistance relationships for each stimulus are approximately parallel to each other. The threshold concentration for both changes is around 0.02 M NaCI. Exposure of the mucosa to stimulation as high as 1 M elicited a relatively small response with high resistance compared with those responses produced by stimulation lower than 0.5 M NaCl. This indicates there is a limiting concentration in tissue function. These relationships approximately coincide with the intracellular studies of frog taste cells (Sato and Beidler, 1975; Akaike et al., 1976). Change in response potential dtflerence

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The NaCl responses in the dorsal epithelium changed in magnitude at various resting potential

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1 min Fig. 5. Effects on the 0.1 M NaCl responses by varying the resting potential difference applied as steady currents through the tissue. Numbers labelled on the left side of each record show the resting potential polarized on the mucosa to grounded serosa.

differences across the tissue. The resting potentials could be changed on the positive mucosa by passing a lo-’ to lo-’ A constant current through the tissue from the mucosa to the serosa and vice versa. Figure 5 shows an example series of the 0.1 M NaCl responses with a variety of the resting potentials. In the middle records labelled 0 mV in the figure, no extrinsic current was applied, the response being (mv)

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5.5 mV. It is seen that the response magnitudes were gradually depressed with a decreasing resting potential on the mucosa and that the polarity of the responses was finally reversed when the resting potential reached below -30 mV. Conversely, increasing the resting potentials caused augmented responses. Figure 6, which was plotted from the records found in Fig. 5, shows the relationship between the resting potential level on the mucosa and the magnitude of the responses to 0.1 and 0.2 M NaCl. Comparing the two curves, it can be seen that the response magnitudes for each resting level are very approximately parallel to each other. The reversal potential of the responses was around -35 mV for a 0.2 M NaCl response and -25 mV for a 0.1 M NaCl response. The same profile of the reversal potential has been observed in the taste cells of frogs and toads (Sato and Beidler, 1975; Akaike et al., 1976; Eyzaguirre et nl., 1972). Effcts of metabolic tetrodotoxin

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An active transepithelial ion transport is known in the dog tongue and other epithelia (DeSimone et al,, 1981, 1984; Ussing and Zerahn, 1951; Herrera, 1966). It also exists in the bullfrog skin and bladder membrane from the data in this study, The NaCl response in these tissues rapidly decreased under the effects of 1 mM ouabain or 2+dinitrophenol (DNE) to the serosa, being associated with marked reduction in the resting potential difference. After about 1 hr, both the NaCl response and the resting potentials in the tissues had decayed to zero, on average. In contrast, in the dorsal epithelium of the bullfrog tongue, neither the magnitude of the responses nor the resting potential difference were influenced when the air supply to the Ringer’s solution was turned off, or 1 mM ouabain was applied to the serosa and/or the mucosa for over 1 hr. When 1 mM DNP was applied to the serosa, the resting potential on the mucosa was found to shift a few mV in a positive direction, but the NaCl response remained unchanged. 4

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I+20 Fig. 6. The relationships between maximum magnitude of the NaCl responses (ordinate) and the resting potential on the mucosa (abscissa). Open and solid circles and their vertical bars represent the responses to 0.1 and 0.2 M NaCl and their standard deviations, respectively. Each circle is derived from four experiments.

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Fig. 7. The time course of depressant effects on the responses to 0.2 M NaCl during an application of 0.01-0.20/, tetracaine on the mucosa. The direction of the arrows indicate the beginning (down) and withdrawal (up) of tetracaine application. The number on each curve represents the concentration of tetracaine applied to the mucosa. The relative magnitude of the response on the ordinate is 100% before applying any tetracaine.

NaCl

response across tongue epithelium

The NaCl response was reversibly depressed in the presence of 0.01-0.2% tetracaine on the mucosa, regardless of no change in the resting potential level. Figure 7 represents the time course of the depressant effects on NaCl responses after various concentrations of tetracaine were applied to the mucosa. The depressant effect on the responses resembled the effects of procaine on the receptor potentials and neural activity in frogs (Akaike and Sato, 1975). However, in comparing the depressant effect of these two local anesthetics on NaCl responses, 5 to 10 times more procaine was required to have the same effect as tetracaine. Tetrodotoxin (TTX) is known to be a selectively powerful blocker for Na activation in excitable cells (Narahashi et al., 1964). However, even after 1 hr of exposure of the mucosa to 10e6 to 10T5 g/ml of TTX, there was still no depression of the 0.2 M NaCl response. There was also no change in the resting potential level. The same results had been observed in the taste cells in frogs (Ozeki and Noma, 1972). Effects of Na or Cl ions freedfrom Ringer’s solution After the dorsal epithelium on both surfaces had adapted to an Na-free Ringer’s solution, substituting NaCl with equimolar choline (choline solution) or tetramethylammonium chloride (TMA solution) caused a greatly decreased NaCl response. After exposure to the choline and TMA solutions for 1 hr, the mean values of the NaCl responses, obtained from 4-6 tissues, were changed to 36.5 and 53.9% in response magnitude, respectively, from the control value of 100%. In contrast, the NaCl response was increased in tissues adapted to a Cl-free Ringer’s solution, substituting NaCl for NaNO, (nitrate solution). There was, on the average, a 120% increase with the nitrate solution for 1 hr.

DISCUSSION

It is well known that, in general, isolated tissue covered with mucosal epithelium, when it is in contact with a physiological solution on both surfaces, produces a transepithelial potential difference across the tissue, the mucosa being negative to the serosa. However, the results in the present experiment showed almost no potential difference across the dorsal epithelium because of the individual polarity, values and standard deviation. On the other hand, there was a large potential difference across the skin and the urinary bladder. These findings suggest that active ion transport in the tissue from bullfrog tongue is ineffective compared to the transport in frog skin (Ussing and Zerahn, 1951) and toad bladder (Herrera, 1966). According to the physiological significance of the tissue, frogs living in water must keep their mouths closed not only to protect the airways and lungs from being filled with water, suggested by the water response in frogs (Zotterman, 1949), but also retain minerals in their bodies. Different results have been reported for the dog tongue (DeSimone er al., 1981, 1984). This could be due to species difference; that is, isolated amphibian tissue is generally insensitive to oxygen and tem-

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perature and is therefore easier than mammalian tissue to keep alive. The NaCl response is not simply a physicochemical potential change occurring at fluid interfaces, because the responses were reversibly depressed in the presence of local anesthetics (Fig. 7) and no potential change was elicited when the serosa was stimulated. The profile of the NaCl response was in many respects similar to the profile of the receptor potential in taste cells and of the taste fibre activity (Sato and Beidler, 1975; Akaike et al., 1976). Therefore, it seems that the NaCl response is an electrophysiological potential change across the dorsal epithelium, including taste organs and may contribute to taste sensation. Taste organs scattered on the examined dorsal surface had diameters of 161 pm on the average, and the area they occupied was only about 5.15% of the total surface area. Furthermore, the ratio of microvilli protruding from the taste cells in the taste organs to the total area of the tongue is extremely small, based on electron microscopic examination of single taste organs of frogs (Dining and Andres, 1976; Toyoshima et al., 1984). Therefore, it is hard to explain how the NaCl response is produced, originating from the receptor potential in the taste cells, even if there is positive feedback via electrotonic coupling between the taste cells and their surrounding cells on the dorsal surface, as has been suggested in the case of non-taste cells in the mud puppy (West and Bernard, 1978). When Eyzaguirre et al. (1972) recorded the intracellular potential from cells of isolated toad tongue mucosa, all surface cells regardless of location responded to taste stimuli. These results raised questions about the degree of specialization of the taste organ cells, and suggested that nonkeratinized lingual cells might contribute to gustation, at least in some species. Thus, a more reasonable mechanism for producing the NaCl response can be explained on the basis of the present experimental results. When NaCl is applied to the mucosa of the bullfrog tongue, the dorsal epithelium is easily penetrated by some ions; these are probably Na ions, because there is no keratinized layer covering the tissue (Fig. 1). Therefore, a transepithelial potential change is produced across the tissue and synchronized with the receptor potential in a large number of taste cells in the organs, resulting in a sufficient field potential on the dorsal surface to affect the input of the intragemmal taste fibres. The change in resistance during the NaCl response and the existence of a reversal potential in the response support this explanation (Figs 3-6). Although the depression of the NaCl response in the tissue, adapted to Na-free solutions, should be expected due to a decrease number of Na ions penetrating it, the augmentation of the response in Cl-free solutions is puzzling. It may be that Na ions are accelerated through the tissue in the Cl-free solution. The finding that TI’X has no influence on the NaCl response also makes it difficult to explain why Na ions penetrate the tissue. This may indicate that TTX cannot penetrate the epithelial intercellular spaces nor affect the NaCl response, as has been suggested by Ozeki and Noma (1972). There have been different findings in the dog

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tongue (DeSimone et al., 1981, 1984). Contrary to our findings in bullfrog tongue epithelium, hypertonic NaCl on the mucosa actively transports ions, producing an increased potential difference across the lingual epithelium. However, based on the evolutionary physiology of the dorsal tongue epithelium, the frog living under water is different from mammals living on land. In fact, the dorsal surface of the bullfrog tongue is flabby to the touch compared with the dog and rat tongue surfaces. Thus, the NaCl response on the bullfrog tongue may contribute to the production or modification of taste sensation. Further analysis of the response may lead to a better understanding of its role. SUMhlARY

1. The electrophysiological characteristics of the dorsal epithelium in the bullfrog tongue were examined. 2. The isolated epithelium in contact with Ringer’s solution on both surfaces produced a negligible potential difference across the tissue, compared with those of skin and bladder. 3. When NaCl was applied to the mucosa, the tissue produced a change in the potential difference so that the mucosa was negative to the serosa (NaCl response). 4. The NaCl response was associated with a decrease in tissue resistance. Both changes in response and resistance depended on stimulus strength. 5. The NaCl response was augmented when the mucosa was polarized positively; whereas it was depressed or reversed under a negative polarity. 6. The NaCl response was not influenced by metabolic inhibitors and tetrodotoxin, but was depressed by local anesthetics. 7. After the tissue had adapted to an Na-free solution, the NaCl response was depressed, but it was augmented in a Cl-free solution. 8. According to the results, the NaCl response may be produced by a passive transport of some ions through the tissue and may contribute to the process of taste reception. Acknowledgement-We would like to thank Dr Toshihide Toh for his histological procedures.

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DeSimone J. A., Heck G. L. and DeSimone S. K. (1981) Active ion transport in dog tongue: a possible role in taste. Science 214 103%1041. DeSimone J. A.. Heck G. L., Mierson S. and DeSimone S. K. (1984) The active ion in transport properties of canine lingual epithelia in oitro. J. gen. Physiol. 83, 633-656.

Dilring M. v. and Andres K. H. (1976) The ulstrastructure of taste organ. CeN Tiss. Res. 165, 181-198. Eyzaguirre C., Fidone S. and Zapata P. (1972) Membrane -potentials recorded from the mucosa of toad’s tongue durine chemical stimulation. J. Physiol. 221, 515-532. Hayash: H. (1978) Rapid penetration of potassium and other salts into the frog tongue papilla. Jap. J. Physiol. 28, 33-45.

Herrera F. C. (1966) Action of ouabain on sodium transport in the toad urinary bladder. Am. J. Physiol. 210,98&986. Kamo N., Miyake M., Kurihara K. and Kobatake K. (1974) Physicochemical studies of taste receptor. II. Possible mechanism of generation of taste receptor potential induced by salt stimuli. Biochem. biophys. Acta 367, 1l-23. Kimura K. and Beidler L. M. (1961) Microelectrode study of taste receptor of rat and hamster. J. cell. camp. Physiol. 5% 131-140.

Mistretta C. M. (1971) Permeability of tongue epithelium and its relation to taste. Am. J. Physiol. 220, 1162-l 167. Murray R. G. (1971) Ultrastructure of taste reception. In Handbook of Sensory Physiology. Chemical Senses Tasfe. Vol. 4, pp. 31-50. Springer, New York.

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Narahashi T., Moore J. W. and Scott W. R. (1964) Tetrodotoxin blockage of sodium conductance increase in lobster giant axons. J. gen. Physiol. 47, 964-974. Ozeki M. and Sato M. (1972) Responses of gustatory cells in the tongue of rat to stimuli representing four taste qualitites. Comp. Biochem. Physiol. 41A, 391-407. Ozeki M. and Noma A. (1972) The action of tetrodotoxin, procaine and acetylcholine on gustatory receptions in frog and rat. Jap. J. Physiol. 22, 467-475. Sato T. (1969) The response of frog taste cells (Rana nigromaculata and Rana catesbeiana). Experientia 25, 709-710.

Sato T. and Beidler L. M. (1975) Membrane resistance change of the frog taste cells in response to water and NaCl. J. gen. Physiol. 66, 735763. Soeda H. and Sakudo F. (1985) Electrical responses to taste chemicals across the dorsal epithelium of bullfrog tongue. Experientia. 41, 5&5 1. Taglietti V., Maffini S. and Casella C. (1971) The recovery cycle of gustatory fibres during chemical stimulation of the tongue. Arch. Sci. biol. 55, 155-164. Toyoshima K., Honda E., Nakahara S. and Shimamoto A. (1984) Ultrastructural and’ histochemical changes in frog taste organ following denervation. Arch. histl. jap. 47, 3142.

Ussing H. H. and Zerhan K. (1951) Active transport of sodium as the source of electric current in short-circuited isolated skin. Acta physiol. stand. 23, 110-127. West C. K. and Bernard R. A. (1978) Intracellular characteristics and responses of taste bud and lingual cells of the mudpuppy. J. gen. Physiol. 72, 305-326. Zotterman Y. (1949) The response of the frog’s taste fibres to the application of pure water. Acta physiol. stand. 20, 181-189.