Responses of the frog gustatory receptors to various odorants

Comp Btochem Ph)s~ol, 1977. to/. 56C. pp 11)5 to I()8 Perqamml Pre.~s. Printed In Great Britain

RESPONSES OF THE FROG GUSTATORY RECEPTORS TO VARIOUS ODORANTS TAI)ASHI KASHIWAG.URA,NAOKI KAMO, KENZO Kt)RIIIARA AND YONOSUKE KOBATAKE Faculty of Pharmaccutieal Sciences, Hokkaido University. Sapporo, Japan {Received 16 June 1976)

1. The frog glossopharyngcal nerve was found to resvxmd to various "'odorants" applied to the tongue. 2. Linear relation held between IogC,h and IogT, where Cth and T stand for the threshold of odorants in thc frog glossopharyngeal nerve response and the olfactory threshold in human, respectively. 3. The effect of the cross adaptation between odorants was examined. The results obtained indicated that the frog gustatory receptor discriminates various odorants. Abstract

INTRODU(.TION In higher vertebrates, olfactory reception takes place at the olfactory cell, which is the terminal swelling of the olfactory nerve. The elicitation of neural responses caused by odorants is not restricted to the olfactory cell but other excitable systems respond to odorants (Bcidler, 1965; Arvanitaki et al., 1967: Tucker, 1971 ; Dethier, 1972). All these systems, which are known to respond to odorants, are the nervus terminalis system. The taste cell of a vertebrate is a secondary sensory cell and its origin is not the nerve cell but the epithelial cell of the tongue. It seems to be widely considered that the secondary sensory cell is much highly differentiated and the taste cell responds only to chemical stimuli of four fundamental taste qualities. In fact, few reports indicating that the gustatory receptors of vertebrates respond to chemicals besides usual taste stimuli have appeared. In the present paper, we report that the frog gustatory receptors respond to various kinds of "odorants". The response to odorants is recorded by measuring the activity of the glossopharyngeal nerve of the frog and the results are discussed in connection with olfactory response in the nose. MATERIALS AND ME'I'HODS Adult bullfrogs. Ranu catc,sbeiana, were used in the present study. The preparation of the tongue with the glossopharyngeal nerve and the recording of nerve activity were carried out by the method described previously (Kashiwagura et al., 1976), which was essentially the ,same as that employed by Kusano (1958). Thc nerve impulses were amplified with a CR-amplificr (lwatsu Electric Co. Type HP-6011, displayed on an nscilloscopc and recorded with a tape recorder (Sony Magncscale Inc. Type FR-3215). The amplified impulses were also integrated with an electronic integrator (Nihon Koden Co. Type RFJ-5) with time constant of 0.3 sec. and the results were recorded with a penwriting recorder. Before application of the stimulating solution, 120 ml of Ringer solution was flowed on the tongue for 2 min (a flow rate of 1 ml/sccJ. Stimulating solutions were applied to the tongue with the same flow rate. The touch artifact 105

was negligibly small compared with the response to chemieal stimulation since the application of adapting solution with the flow rate to the tongue did not produce any response. After each application of the stimulating solution on the tongue surface, the tongue was rinsed with Ringer solution. About 15 min were interposed between each successive stimulation unless otherwise noted. All experiments were performed at 1TC. The chemicals used in the present study were analytical grade without further purification. Odorants were dissolved in Ringer solution. The composition of Ringer solution was: 112 mM NaCI, 3.4 mM KCI, 2.5 mM CaCI 2, and adjusted pH to 7.1 with 2.5mM NaH('O3-HCI buffer.

RES[:LTS AND DISCI:K~;ION The frog glossopharyngeal nerve responded to various odorants applied to the tongue (The records of the integrated response arc illustrated in Fig. 3.). The responses to relatively low concentration of odorants were phasic type in most cases and a tonic component often appeared in the responses induced by odorants of relatively high concentration compared with the threshold for respective odorants. Among various odorants used in the present study, ethanol was the only substance that other investigators (Diamant et at., 1963; Diamant et al., 1965: Hcllekant, 1967) had applied to the gustatory organs. They found that ethanol induced the neural response in the chorda tympani nerves of the cat, dog and rat. Hellekant (1967) stated that a slow onset of the discharge of the rat chorda tympani nerve was observed initially during application of ethanol to the tongue. The application of various odorants as well as ethanol brought about a rapid onset of thc disehargc of the frog glossopharyngeal nerve (see Fig. 3) as is similar to the cases of stimulation by usual taste stimuli. The fact that the response appeared instantly with application of odorants, together with the fact that chemicals hardly penetrate the tongue epithelium (Mistretta, 1971) suggests that the response of the glossopharyngeal nerve is not induced by direct stimulation of the nerve but stimulation of the gustatory receptors by odorants.

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Fig. I. Magnitude of the integrated responses of the frog glossophaDngeal nerve as a ftmction o l odorant concentration. The magnitude of the response to 3 mM ('aCI 2 is taken as a unit of the ordinate. In Fig. 1. tile magnitude of the initial peak of the integrated responsc is plotted against concentration of odorants. In the ordinatc of the figt, rc, the magnitude of thc response to 3 mM CaCI 2. which induces a large responsc in the frog glossopharyngeal ncrvc. is takcn as a unit. The threshold concentration of odorants to elicit the response varies cxtensively with odorant species applicd. In Fig. 2. the threshold concentration (Cu,) of the odorants for thc frog glossopharyngcal nerve, which were taken from thc data shown in Fig. 1. is compared with olfactory thrcshold (I) for human. The values of T, which wcrc taken from thc literature (Koyama & Kurihara. 1972). arc represented in numbcr of moleculcs of odorant per ml of the air. As seen from thc tigure, plots of logT against Iog(',h gavc a straight line. A similar linear rclation was obtained between olfactory threshold in human and 16

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the concentration of odorants giving a surface pressure increase of 1.0 dyne:'cm in tile monolavcrs, of lipids extracted from bovine olfactory epithelitma (Koyama & Kurihara, 1972). These results together with the fact that the odorants are generally lipoid soh, blc suggcstcd that the olfactory stimulation is induced by adsorption of adorants on the hydrophobic parts of the receptor membrane. The relation shmx.n in Fig. 2 can also bc undcrstood provided that the response of the frog glossopharyngeal nerve to odorants is induced by a similar mcchanism. It v induce approximately equal magnitude of the response. As seen from the tigurc, the responses to methanol and I-propanol x~crc almost suppressed b3 the adaptation of the gustatory receptors to ethanol. On the other hand. the magnitudc of the response to l-butanol was pratically unchanged before and after application of ethanol: the gustatory ncrvc responded to l-butanol even after the gustatory receptors bec'amc insensitive to ethanol. The response to 1-octanol also appeared aftcr adaptation to ethanol. The above results suggcst that thc cross adaptation is seen only between the pairs of alcohols whose carbon chain length is close to each other. l-igurc 3b illustrates the records obtained ~ith the pairs of isoam',l acetate ethyl hntyrate, isoarn3.1 acctatc.-coumarin and iszmmyl acetate l-octanol. The response to ethyl bt, tyratc was practically suppressed after application of isoamyl acetate but the responses to coun-larin and l-octanol were not grcatb arfcctcd. Figure 3c illt, strates the records obtained with the pairs of mcnthone eth,,l ether, ethanol isoamyl ace-

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Fig 3a, b.c. Integrated response of the glossopharyngeal nerve to various pairs of odorants. The experiment for A in the figure, for instance, was carried out as follows; methanol was applied and washed with Ringer solution. After about 10 min, ethanol and subsequently methanol were applied. A: 500 mM methanol, 3(X)mM ethanol, B: 100mM l-propanol, 300mM ethanol. C: 200mM l-butanol, 300mM ethanol. D: l mM ethyl butyrate, l mM isoamyl acetate, E: 2 mM coumarin, l mM isoamyl acetate, 1-: 0.5raM l-octanol, l mM isoamyl acetate. G: 50mM ethyl ether, 0.2mM menthone. H: I m M isoamyl acetate. 300 mM ethanol, I: 2 mM coumarin, 50 mM ethyl ether.

tate and ethyl ether coumarin. No cross adaptation effect was observed in the first two cases. It is hard to obtain systematic conclusion in connection with quality of odor since there are n u m e r o u s o d o r a n t s and the classification of o d o r a n t s is not always established. However, we can say at least that the frog gustatory receptors discriminate various odorants in

a sense that the receptors respond to other odorants even after the receptors became insensitive to one odorant. It is worth noting that the frog gustatory receptors, which is considered to have no specific receptor molecule for odorants, can discriminate various odorants in the sense described above, although there is no evidence indicating that the

TADASIII KASItlWAGURAel ~ll.

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mechanism of discrimination of various odorants at the gustatory receptors is similar to that at the olfactory receptors in the nose at the present.

SUM.MARY 1. Frog glossopharyngeal nerve was found to respond to various odorants applied to the tongue, and the relation between the magnitude of the response and odorant concentration was examined with l0 spcties of odorants. The threshold concentration to elicit the response varied extensively with odorant species. 2. Plots of IogCth against logT gave a straight line, where C,h and T stand for the thrcshold of odorants in the frog glossopharyngeal nerve response and the olfactory threshold in human, respectively. 3. The effect of the cross adaptation between odorants was examined with various pairs of odorants. it was found with many pairs that the gustatory receptors responded well to other odorants even after the rcceptors became inscnsitive to one odorant by the self adaptation. This indicated that the gustatory receptors can discriminate various odorants in this sense.

RFFERENCI'LS ARAVANITAKIA., TAKIU('HI H. 8¢, CIIAI,AZONITIS N. (1967) Specific unitary osmorcccptor potentials and spiking patterns from giant ner,,e cells. In O!/~wtion and Taste (Edited b) HA~ASHt T.I VoI. 2. pp. 573-598. Pergamon Press, Oxford.

BHDHiR I.. M. (1965) ('omparison of gustatory receptors. olfactory receptors, and free nerve endings. (old Spring llarb. Syrup. Quant. Biol. 30, 191 2(X). DErHIER V. G. (i972) Sensitivity of contact chemoreceptors of blowfly to vapors. Proc. :",:atn Acad. Sci. U.S.A. 69, 2189-2192. DIAMAN'r H.. FUNAKOSIII M., SIR()M L. & ZOTTF,RMAN Y. (1963} Electrophysiological studies on human taste nerves. In Offaction and Taste (Edited b) ZO'I'rERMAN Y.) Vol. 1. pp. 193 203. Pergamon Press, Oxford. DIA.Vu~.NT H., FUNAKOSIII M., SI'ROM L., WELLS C ~¢, ZOTTERMAN Y. (1965) A comparison of neural and psycophysical responses to taste stimuli in man. Acta Physiol. Stand. 64. 67-74. HELLEKAYT G (1967} Action and interaction of ethyl alcohol and some other substances on the receptors of the tongue. In O!faction and Taste (Edited by ttAYASttl T.) Vol. 2. pp. 465-479. Pergamon Press. Oxford. KASHIWA(WRA T., KAMO N.. Kt'RIHARA K. & KOBAIAKE Y. 11976) Phasic and tonic components of gustatory response in thc frog. A m J. Physiol. (In press). KOVAMA N. & Kt:mHARA K. (1972) Effcct of odorants on lipid monolaycrs from bovine olfactory epithelium. Nature. l.ond 236. 402 404. KCSANO K. (1958)The influence of cations on the activity of gustatory receptors I. effects of KCI. Kumamoto Med. J. 11, 174 183. MlSrREI IA C. M. ( 19711 Pcrmcabilit.v of tongue epithelium and its relation to taste..4m. J. Physiol. 220, 1162-1167. SMtm D. V. & FRAYK M. (1972) Cross adaptation between ~dts in the chorda tympani nerve of the rat. Physiol. Behav. 8, 213 220. TVCKtiR D. (1971) Nonolfactory responses from the nasal cavity: Jacobsons organ and the trigeminal system. In llandhook qfSensory Physiol. (Edited by BEU)L[:R, L. M.) Vol. 4-1, pp. 151 181. Springer. Ncw York.