0-01m saccharin buffered with tris(hydroxymethyl)aminometh

J. Physiol. (1969), 204, pp. 311-329 311With 8 text-figuresPrinted in Great Britain

GUSTATORY IMPULSE DISCHARGES IN RESPONSE TOSACCHARIN IN RATS AND HAMSTERS

BY H. OGAWA, M. SATO AND S. YAMASHITAFrom the Department of Physiology, KumamotoUniversity Medical School, Kumamoto, Japan

(Received 31 October 1968)

SUMMARY

1. Impulse discharges produced by saccharin sodium as well as four basicgustatory stimuli were recorded in chorda tympani fibres of rats andhamsters.

2. Units predominantly sensitive to NaCl showed a concentration-response magnitude curve for saccharin sodium similar in shape to that forNaCl but smaller in magnitude. Units of this category did not respond to0-01 M saccharin buffered with tris(hydroxymethyl)aminomethane.Response to saccharin sodium in these units are considered to result fromstimulation of receptor molecules by Na ions.

3. Units predominantly sensitive to sucrose showed a concentration-response magnitude curve for saccharin sodium with a maximum magni-tude at about 0-01-0-1 M. Impulse discharges produced by sucrose andsaccharin sodium showed rhythmic burst-like firing. Units of this categoryresponded well to 0-01 M saccharin. Responses to saccharin sodium in theseunits are attributed to the reaction between saccharin molecules andreceptor sites.

4. The optimum concentration at which a greatest response magnitudewas found varies from one unit to the other and is inversely related tosucrose sensitivity, units highly sensitive to sucrose showing a low optimalconcentration. Presence of the optimum concentration is explained by amechanism known as the non-competitive auto-inhibition.

5. Off-responses were observed in units predominantly sensitive tosucrose when 003-1 M saccharin sodium applied to the tongue was rinsedwith water.

6. Neural information for saccharin sodium is described quantitativelyin relation to that for four basic gustatory stimuli.

H. OGAWA, M. SATO AND S. YAMASHITA

INTRODUCTION

In a pioneering work Pfaffmann (1941) first demonstrated that gustatorynerve fibres mediate information for more than one kind of chemicalrather than for one of those representing the classical four basic gustatoryqualities. This has been confirmed later by several investigators on rats,cats, hamsters and rabbits (Pfaffmann, 1955; Cohen, Hagiwara & Zotter-man, 1955; Fishman, 1957; Nagaki, Yamashita & Sato, 1964), and abroader multiple sensitivity of chorda tympani fibres for a variety ofchemicals was found, although it has often been pointed out that somefibres respond relatively well to a particular kind of stimulus and otherfibres are more sensitive to another kind of stimulus (Pfaffmann, 1955).Fishman (1957) reported in his study on single unit responses of rats andhamsters that there are two categories of units, which differ from eachother in their sensitivity to sucrose and a salt and in their concentration-response curve for saccharin sodium.An attempt to classify chorda tympani fibres of rats and hamsters

according to their responsiveness to four basic gustatory stimuli andthermal changes has recently been made by Ogawa, Sato & Yamashita(1968), who demonstrated several categories of units, which differ fromone another in their sensitivity to gustatory and thermal stimuli as well asin their spontaneous discharges. They showed further that, in the hamster,units predominantly sensitive to sucrose are sharply differentiated fromthose responding to NaCl, although the distinction is not so marked in ratsas in hamsters.In the experiments reported here, responses of chorda tympani fibres of

both rats and hamsters to saccharin sodium were recorded. Difference inthe response to saccharin sodium was investigated between units belongingto different categories, and the mechanism of the action of saccharinsodium on gustatory receptors was analysed. Finally, neural coding fortaste of saccharin sodium was described in relation to neural informationfor four qualities of taste.

METHODS

Impulse discharges in response to stimulation of the tongue by saccharin sodiumas well as the four basic stimuli (0.1 m-NaCl, 0-5 M sucrose, 0.01 N-HCl and 0-02 Mquinine hydrochloride), which had been recorded from fifty chorda tympani fibresof rats and twenty-eight fibres of hamsters in the experiments described in an earlierpaper, were used mainly for the analysis. Concentration-response magnitude re-lationships for saccharin sodium, sucrose and NaCl were examined on twelve fibresof rats and six fibres of hamsters. Additionally, impulse discharges produced by0 02 M saccharin sodium as well as the four basic stimuli were recorded in twenty-seven fibres of rats, and on several of these fibres effects of 0 3 M saccharin sodium,0.01 M saccharin, 1 M glucose, 0 005 M dulcin and 0 3 M monosodium glutamate were

312

GUSTATORY RESPONSE TO SACCHARINexamined. When 0 01 M saccharin, which is nearly the maximum soluble concen-tration, was used as a test stimulus, it was buffered to pH 4-8 with 0*01 M tris(hydroxymethyl) aminomethane, because an 0 01 M solution of saccharin had pH of2-2.Numbers of impulses discharged in the first and second 5 see periods after stimu-

lation were counted, and used to represent the magnitude of response to variouschemicals. No subtraction of the number of spontaneously discharged impulses wasmade from that of impulses discharged in response to stimuli, because the former wassmall. To check the variability of responses, several repeated series of stimulationsby five kinds of chemicals were carried out on two chorda tympani fibres of rats.The mean and S.D. of responses to each stimulus is represented in Table 1, whichshows that responses to HCl and quinine appear to be somewhat variable comparedwith those to other stimuli.

Computation of equations for neural data was made in the Kumamoto UniversityComputation Centre.

TABLE 1. Number of impulses discharged in the first and next 5 seeafter stimulation by five chemicals

Fibre Stimuli

No. 15 0.1 M-NaCl0-5 M sucrose0.01 N-HCl0U02 M quinine hydro-chloride002 M saccharin sodium

No. 16 0 1 M-NaC10-5 M sucrose0-01 N-HCl0-02 M quinine hydro-chloride

0-02 M saccharin sodium

Impulse numberin the

initial 5 see

400± 5*97 (6)4-0± 1-79 (5)79-6+ 19-82 (6)43.5 ± 8-65 (6)

9-0 + 1*79 (5)

102-5 ± 15-41 (6)1.1 + 1j51 (6)

28-3 + 14-51 (6)7.7 ± 5-03 (6)

43-0 + 5 07 (6)

Impulse numberin the

next 5 see

17-7 + 6-58 (6)4-0 + 2-00 (5)

50.5 + 11*68 (6)21-7 + 5-68 (6)

7 0 + 1-41 (5)

59-1 + 2097 (6)1.0+±182 (6)

20-7 + 10-04 (6)4-3 ± 2-77 (6)

18-0 + 2-77 (6)

Numerals are the mean + S.D. of impulse numbers discharged in response to re-peated stimulations, while those in the parentheses indicate number of trials.

RESULTS

Response profiles of chorda tympanifibres for saccharin sodium. A majorityof chorda tympani nerve fibres of rats and hamsters was found to respondto 0-02 M saccharin sodium to varying degrees. This is shown in Fig. 1, inwhich the response profile for 0-02 M saccharin sodium as well as four basicgustatory stimuli of twenty-eight hamster chorda tympani fibres is pre-sented. As shown in a preceding paper (Ogawa et al. 1968), hamster chordatympani fibres are divided into two major categories, the one being pre-dominantly sensitive to sucrose and the other sensitive to NaCl. Some ofthe latter fibres responded to quinine and HCl as well. Units predomin-

313

314 H. OGAWA, M. SATO AND S. YAMASHITAantly sensitive to sucrose gave good responses to 0-02 M saccharin sodium,responsiveness for the latter being in parallel to that for the former (Fig. 1).In some units sensitive to NaCi, 002 M saccharin sodium also producedimpulse discharges, the amount of which was in parallel to that of re-sponses to 0.1 M-NaCl (Fig. 1). A similar situation can also be recognizedin the response profiles of rat chorda tympani fibres shown in Fig. 2. Itis seen in Figs. 1 and 2 that the responses to 002 M saccharin sodium in

HamsterSaccharin sodium 150

NaCI 200

100

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Es Sucrose100

HC1100

Quinine

NOPQRSTU VXYZ abABCDE FGH I JK LMChorda tympani fibres

Fig. 1. Response proffle of twenty-seven chorda tympani fibres of hamstersfor 0-02 m saccharin sodium, O01Im-NaCl, 0-5 m sucrose, 0-01 N-HC1 and0-02 m quinine hydrochloride. Fourteen fibres are arranged in the order ofresponsiveness for 05 M sucrose and the remaining thirteen fibres in theorder of responsiveness for 0.1 M-NaCl. Ordinate represents the number ofimpulses discharged in the first 5 see after stimulation, while abscissaindicates individual nerve fibre.

GUSTATORY RESPONSE TO SACCHARIN

units sensitive to sucrose are nearly the same as those to 0 5 M sucrose,but that the responses produced by 0-02 M saccharin sodium in NaCl-sensitive units are smaller than those to 0-1 M-NaCl. The latter fact maybe due simply to a low concentration of saccharin sodium because fourteen

Rat Saccharin sodium

100

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100K Quinine

indciuswkrXNABCDEFGHIJKLMOPQRSTUVYZabefghjImopqtChorda tympani fibres

Fig. 2. Response profile of forty-seven chorda tympani fibres of rat for 0-02M saccharin sodium, 0 1 M-NaCl, 0 5 M sucrose, 0 01 N-HC1 and 0 02 M qui-nine hydrochloride. Eleven fibres are arranged in the order of responsive-ness for 0-5 M sucrose, and the remaining thirty-six fibres in the order ofresponsiveness for 0-1 M-NaCl. Ordinate represents the number of impulsesdischarged in the first 5 sec after stimulation, abscissa, the individual nervefibres.

fibres out of seventeen, on which the effect of 0-3 M saccharin sodium wasexamined, showed greater sensitivity to 0.1 m-NaCl than that to 0-5 Msucrose and gave responses to 0-3 M saccharin sodium, the amount of whichbeing nearly of the same order of magnitude as that for 0-1 M-NaCl.

315


The number of impulses discharged by 0-02 M saccharin sodium wasplotted in Fig. 3 as a function of that elicited by 0017 M-NaCl. The relation-ship between the response to saccharin sodium and that to NaCL is ex-pressed approximately by a straight line having a slope of 0 97 for twelveunits which showed high sensitivity to NaCl and poor sensitivity to sucrose.On the other hand, four units responding well to sucrose showed highsensitivity to saccharin sodium, and their magnitudes of responses tosaccharin sodium deviated from the straight line relationship to the

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Response to 0 017M-NaCI (impulses/5 sec)Fig. 3. Relationship between the number of impulses discharged by 0*017M-NaCl and that by 0-02 M saccharin sodium in sixteen chorda tympanifibres of rats.Numerals beside each point indicate number of impulses discharged by

0-5 M sucrose. The straight line shows the regression equation of the magni-tude of response to saccharin sodium on that for NaCl, y = 0 97x - 1*08(r = 0.84) in twelve units predominantly sensitive to NaCl (filled circles).Open circles represent units responding to 0 5 M sucrose better than to 0.1M-NaCl. S.D. of y = 10-97.

magnitude of NaCl responses. The presence of a straight line relationshipbetween the response magnitude for saccharin sodium and that for NaClsuggests that sodium ions are the stimulating factor in eliciting responsesto saccharin sodium in units predominantly sensitive to NaCl. On theother hand, a sharp differentiation of sucrose-sensitive units from NaCl-

316

GUSTATORY RESPONSE TO SACCHARINsensitive units suggests that the anion portion of saccharin sodium excitesunits predominantly sensitive to sucrose.

Selective response of chorda tympani fibres to saccharin. In order to testthe hypothesis that in the response to saccharin sodium the receptorssensitive to NaCi are primarily stimulated by sodium ions and that thoseresponding to sucrose are excited mainly by the anionic portion of sac-charin sodium, the stimulating effect of saccharin was examined. Figure4 represents impulse discharges produced by the four basic stimuli, 0-01 Msaccharin sodium and 0-01 M saccharin in a unit predominantly sensitiveto NaCl but not to sucrose. As seen in Fig. 4, 0-01 M saccharin sodiumgave rise to a moderate response in this unit, whereas saccharin of thesame concentration buffered with Tris was ineffective. A delayed impulsedischarge by 0-01 M saccharin may be due to its low pH, because this unitshowed a similar response to HC1 (pH 2.0). On the other hand, both 0-01 Msaccharin sodium and Tris-buffered 0-01 M saccharin (as well as 0-01 Msaccharin) produced impulse discharges in a unit predominantly sensitiveto sucrose but not to NaCl (Fig. 5). A solution of 0-01 M Tris aminomethanedid not elicit any response in this fibre. Only two units predominantlysensitive to sucrose out of eight, on which the effect of saccharin wasexamined, gave good responses to Tris-buffered 0-01 M saccharin while inthe five units primarily sensitive to NaCl no responses were obtained.These results are consistent with the hypothesis that receptor moleculesover the surface of receptors sensitive to NaCl react with Na ions whilethose sensitive to sucrose combine with the anionic portion of saccharin.As shown in Fig. 5, impulse discharges elicited by 0 5 M sucrose and

0-01 M saccharin in a unit predominantly sensitive to sucrose consist ofrhythmic burst-like firing with a fairly constant interval between groups ofimpulses. On the other hand, impulse discharges produced by NaCl orsaccharin sodium in a unit primarily sensitive to NaCl did not showrhythmic burst-like firing (Fig. 4). Impulse discharges produced by sweetsubstances acting on units more sensitive to 0-5 M sucrose than to 0.1 M-NaCl presented a periodicity of 250-750 msec, while units predominantlysensitive to NaCl did not show any periodicity in impulse trains producedby either saccharin sodium or NaCl (Sate,, Yamashita & Ogawa, 1969). Thedifference in the temporal pattern of impulse trains between the responseproduced by sucrose and saccharin sodium in sucrose-sensitive units andthat by NaCl and saccharin sodium in NaCl-sensitive units indicates differ-ence in the mechanisms underlying the reaction between chemical sub-stances and receptor molecules between the two. As suggested already, inthe former case the reaction presumably occurs between sucrose or sac-charin and receptor molecules sensitive to sucrose, while in the latter caseNa ions presumably react with Na+-sensitive receptor molecules.

317


Concentration-response magnitude relationships. Figure 6A showsrelationships between the number of impulses discharged in 5 sec afterstimulation and the concentration of saccharin sodium, sucrose and NaClobtained from two chorda tympani fibres of rats, one predominantly sensi-tive to NaCl and the other more sensitive to sucrose than to NaCl. Bothunits responded to saccharin sodium very well, but there is a marked

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001M saccharin 1111 I 1

sodium W2-BII iIII .. -I--1- -- I--n ----'-I

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fl.flM trnt.

Fig. 4. Impulse discharges produced by six kinds of chemicals in a singlerat chorda tympani fibre, predominantly sensitive to NaCl. Note theabsence of response to 0*01 M saccharin solution buffered with Tris. Delayedimpulse discharges in the response to 0*01 M saccharin probably resultedfrom its low pH because of their similarity to those in the responses to HCL.Time signal: 0 1 sec.

01M -NaCI

I_i I .1-- 1 1 AO5ri sucros fsinw~l i"m..-~ _ ;

IT fl [iI ILl 41r4I HI

OO1N-HCI,~~~-l

F .v .,,.,.,.6 ., Ml0-02M quinine

0-3M saccharinsodium

.~~~~~ -

O-0lm saccharin

sodium l.d ,_II Lti Vi I III JI 1PMJ

OO1M saccharin

O01M tris-bufferedsaccharin

--- - - 1-

sec

Fig. 5. Impulse discharges produced by six kinds of chemicals in a singlerat chorda tympani fibre, predominantly sensitive to sucrose. Note re-

sponses to Tris-buffered 0-01 M saccharin.

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318


difference in the stimulus-response relationship for saccharin sodiumbetween the two. In the unit predominantly sensitive to NaCi (filledcircles) the magnitude of the response to saccharin sodium increased withan increase in concentration up to 1 M, the concentration-response magni-tude relationship being approximately similar to that for NaCl. On theother hand, in the unit more sensitive to sucrose than to NaCl (open circles)the magnitude of response to saccharin sodium increased with an increasein concentration up to 003 M, but declined abruptly with a further in-crease in concentration. Out oftwelve units of the rat examined, three weremore sensitive to sucrose than to NaCl, and they showed a bell-shapedconcentration-response magnitude relationship, the threshold concentra-tion being about 0 0003 M and the maximum magnitude of response lyingat 003 M. On the other hand, eight units out of twelve, which were pre-dominantly sensitive to NaCl, showed a monotonically increasing magni-tude of response with increasing concentration, the threshold concentrationranging from 00003 to 0-1 M.

Five hamster units out of six showed a larger response to sucrose thanthat to NaCl, and all the hamster units presented a bell-shaped concen-tration-response magnitude relationship with a maximum at 001-03 M.The relationship obtained from six hamster units are shown in Fig. 6B.The saccharin concentration, at which the maximum magnitude of re-sponse was obtained, varies from one unit to another according to sensi-tivity for sucrose relative to that for NaCl. The situation can be betterillustrated in Fig. 7A, where the saccharin concentration, at which themaximum response magnitude (Rm) had been obtained, was plotted as afunction of the magnitude of response to 05 M sucrose. As can be seen inFig. 7A, units showing a high sensitivity to sucrose presented a maximumresponse at a low saccharin sodium concentration, i.e. in both hamsters andrats the concentration for Rm is inversely related to the magnitude ofresponse to sucrose (1?). The number ofimpulses (Nm) discharged in sucrose-sensitive units at the saccharin concentration for Rm is almost linearlyrelated to the magnitude of response to 0 5 M sucrose, as shown in Fig. 7B.In this figure, in addition, the magnitude of response to 002 M saccharinsodium was plotted as a function of RX, the former being shown to belinearly related to the latter. In the figure three points at the left hand sidedeviate from a linear relationship between Nm and R,. These units arepredominantly sensitive to NaCl and their response to saccharin sodium isdifferent in nature from that in other units primarily sensitive to sucrose.Two units among twelve chorda tympani fibres of rats, in which con-

centration-response magnitude relationship had been examined, wereextremely sensitive to HC1 and quinine and moderately to NaCl but not tosucrose. Responses of one of these units to HCl, quinine, NaCl and sac-

319


charin sodium of varying concentrations are shown in Fig. 8A, whichshowed a very high threshold for saccharin sodium, and the number ofimpulses discharged by 1 M saccharin sodium is small compared with thatproduced maximally by HCl, quinine and NaCl. Several fibres of this cate-gory can be found in Figs. 1 and 2. These results indicate that, at low con-centrations of saccharin sodium, units predominantly sensitive to NaClor to sucrose are stimulated and that only at high concentrations of sac-charin sodium are units sensitive to HCl, quinine and NaCl excited.

A

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100 A 82/1x 53/71

50 A 13/4A

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0*000 0~00 0.1

Saccharin sodium concentration (M)Fig. 6. A. Concentration-response magnitude relationships for saccharinsodium (continuous line), sucrose (dashed line) and NaCl (dashed anddotted line) in two chorda tympani fibres of rats, one predominantlysensitive to sucrose (0; fibre r in Fig. 2) and the other primarily sensitiveto NaCl (0; fibre F in Fig. 2). B. Concentration-response magnituderelationships for saccharine sodium in six chorda tympani fibres ofhamsters.Numerals at the top of the figure indicate numbers ofimpulses discharged inthe first 5 sec after stimulation by 0 5 m sucrose and that for 0.1 M-NaCl inindividual units.

+, 0, 0, A, x and A correspond to fibres b, Y,X, U,F andO in Fig.1respectively.

320

GUSTATORY RESPONSE TO SACCHARINImmediately after saccharin sodium solution had been rinsed off the tongue with

water, sudden impulse discharges (off-response) were observed in a unit pre-dominantly sensitive to sucrose. Concentration-response magnitude relationships foron- and off-responses to saccharin sodium are shown in Fig. 8B. In the Fig. thelowest concentration of saccharin sodium at which the off-response was observed wasfound to be 0-03 M, while the optimum concentration, at which the greatest magnitudeof the off-response was obtained, was 0-3 M. Similarity of shape of the concentration-response magnitude relationship between the on- and off-responses suggests that theoff-response is due to re-stimulation of gustatory receptors, which had been in-activated during flow of concentrated saccharin solution, as a consequence ofdilution of the solution by water. This possibility may additionally be supported bythe fact that the off-response commenced at or near a concentration where the on-response showed a greatest magnitude of response.

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Response to 0-Sm Sucrose (impulses/S sec)Fig. 7. A. Relationship between saccharin sodium concentration, wherethe greatest response magnitude was obtained, and the response magni-tude for 0-5 m sucrose (number of impulses discharged in the initial 5 sec),obtained in chord tympani fibres of hamsters ( 0) and rats ( 0). Numeralnear each point indicates number of impulses discharged in the initial 5 secafter stimulation by 0-1 m-NaCl in individual fibres. B. Relationshipbetween the maximum magnitude of response to saccharin sodium of vary-ing concentrations and the response magnitude for 0-5 m sucrose in chordatympani fibres of hamsters (0) and rats (AL). Numerals near each opencircle and triangle represent concentration for Rm. Filled circles andtriangles represent the magnitude of response to 0-02 M saccharin sodiumin individual units of hamsters ( *) and rats (A), respectively.

In Figs. 6A and 8B, concentration-response magnitude relationshipsfor both sucrose and saccharin sodium in units predominantly sensitive tosucrose are demonstrated. It is readily seen in these figures that the concen-tration of saccharin sodium which produced a response magnitude equalto that for sucrose is about one log unit below the sucrose concentration,i.e. 0-01 M saccharin sodium produced a magnitude of response approxi-mately equal to that by 0-1 M sucrose. In addition, the Rm for saccharin

321

322 H. OGAWA, M. SATO AND S. YAMASHITAsodium is about one or 1-5 log units below the Rm for sucrose. In threeunits of hamsters and two units of rats, for which concentration-responsemagnitude relationships for sucrose and saccharin sodium were examined,the saccharin sodium concentration eliciting a response magnitude equalto that for sucrose was found to be one or 1.5 log units below the sucroseconcentration.

0150 A

A ~~~~~0

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50~~~~~~~~~~~

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00000 01 0-1 1

0

8 150 BE

1000~~~~~~~~~~~0

50~~~~~~~~~~~0

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0.001 0*01 0.1 1Concentration (M)

Fig. 8. A. Concentration-response magnitude relationships for NaCi(0), HC1 (A), quinine (A) and saccharin sodium ( 0) in a chorda tym-pani fibre ofa rat, predominantly sensitive to NaCl, HC1 and quinine (fibrePin Fig. 2). B. Concentration-response magnitude relationships for sucrose(triangles) and saccharin sodium (open circles) in a chorda tympani fibreof a hamster (fibre b in Fig. 1). Filled circles indicate the off-responseappearing after rinse of saccharin solution with water.

As shown in Fig. 6A, in the units predominantly sensitive to NaCl theconcentration-response magnitude relationship for saccharin sodium issimilar in shape to that for NaCl, but, in general, the magnitude of responseto saccharin sodium is slightly less than that for NaCl when their values atan equal concentration are compared with each other. The average numbersof impulses discharged in the initial 5 sec, in six units predominantly sensi-


tive to NaCl, by saccharin sodium and NaCl of 0-01, 003, 0.1, 03 and 1 Mwere 18, 36, 39, 56, 72 and 18, 49, 83, 89, 97, respectively.

Quantitative description of neural information for saccharin sodium. Sincethere are few gustatory fibres which mediate information for only one ofthe four basic gustatory stimuli, it has been proposed by Pfaffmann (1955),Erickson (1963) and Erickson, Doetsch & Marshall (1965) that discri-mination of taste qualities depends on the similarity or dissimilarity ofneural activity pattern across many fibres and that this can be representedquantitatively by a correlation coefficient between a pair of stimuli. There-fore, correlation coefficients were calculated between the amount of re-sponse to saccharin sodium and those for the four basic gustatory stimuli(Ogawa et al. 1968). A significant correlation was found between theamount of responses to 0-02 M saccharin sodium and that for 0 5 M sucrose(r = 0 74 and 0X83, when calculations were based on numbers of impulsesdischarged in the first 5 see after stimulation in twenty-seven hamsterunits and forty-seven rat units, respectively), while a very small corre-lation was obtained between the former and the amount of responses to0X1 m-NaCl (r = 0.10 and 0-13 in the hamster and rat, respectively).Similar results were obtained by calculating correlation coefficients be-tween pairs of stimuli, based on the data of twenty-seven fibres of rats.They are summarized in Table 2, in which 0 3 M saccharin sodium shows ahighly significant correlation with 0-1 M-NaCl but not with 0 5 M sucrose.A high correlation coefficient between 0-02 M saccharin sodium and 0-5 Msucrose indicates similarity of taste quality to each other, while presenceof highly significant correlation between 0 3 M saccharin sodium and 0-1M-NaCl reveals salty taste of the former.

1 M glucose produced in chorda tympani fibres of rats a response comparable inmagnitude to that to 05 m sucrose, the correlation between the two being highlysignificant (Table 2), while 0005 M dulcin elicited little response even in a unit pre-dominantly sensitive to sucrose. This finding is consistent with the fact that ratsprefer saccharin to water but do not prefer dulcin (Fisher, Pfaffmann & Brown, 1965).Monosodium glutamate (MSG), 0'3 M, produced responses very similar in magnitudeto those elicited by 03 M saccharin sodium, the correlation between the two beinghighly significant (Table 2), but the former appears to be positively correlated with0.1 M-NaCl and negatively correlated with 0-02 M quinine, indicating a differencein taste quality from 0 3 M saccharin sodium.

Since Fig. 1 reveals that the amount of response to 0-02 M saccharinsodium (z) is partially correlated with that for 0.1 M-NaCl (x) and for 0 5 Msucrose (y), partial correlation coefficients were calculated between thethree. The partial correlation coefficient obtained from numbers ofimpulsesdischarged during 5 see after stimulation in twenty-seven hamster unitsis 0X67 for z and x and 0-87 for z and y, and the relationship between z, xand y is expressed by a regression equation z = 0 32x + 0-75y - 0-462, the

II Phy. 204

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multiple correlation coefficient being 087. This equation establishes themanner in which neural information for 0-02 M saccharin sodium in ham-sters is extracted from that for 0.1 m-NaCl and that for 0 5 M sucrose.

Extending this concept further, one is able to obtain equations whichexpress the neural information for saccharin sodium by those for fourbasic gustatory stimuli, because, as seen in Figs. 1 and 2, the amount ofresponses is considered to be related to those for four basic stimuli.Equations for 0X02 M and 0 3 M saccharin sodium, obtained by calculatingthe relationships between the amount of responses to saccharin sodiumand those for four basic stimuli according to the least squares method, arepresented in Table 3. Similar calculations were made with the data on 1 Mglucose and 0 3 m-MSG. The equations reveal how the neural informationfor a particular stimulus consists of those for the four basic stimuli andpredict a quality of taste for this stimulus. The coefficient for x, y, u and vmay vary when the concentration of x, y, u or v changes. For example, inthe equation for 0-02 M saccharin sodium, in which x is 041 m-NaCl, thecoefficient for 0 5 M sucrose is largest, but in the equation for the same sub-stance, in which x is 0017 m-NaCl, the coefficients for x and y becomenearly equal to each other.

DISCUSSION

In a preceding paper Ogawa et al. (1968) have shown that responsivenessof chorda tympani fibres of rats and hamsters to a certain pair of gustatorystimuli is not independent from one another, i.e. a certain category ofunits tends to respond to quinine as well as HC1 and that units sensitiveto NaCl have little tendency to respond to sucrose. Especially in thehamster, sucrose sensitivity and NaCl sensitivity showed a significantnegative correlation with each other. In the present paper evidence indi-cating a distinct difference in the concentration-response magnituderelationship for saccharin sodium and in the temporal pattern of impulsedischarge between sucrose-sensitive and NaCl-sensitive units has beenpresented, and the difference has been attributed to a difference in thereceptor molecules over the gustatory receptor membrane and in themechanism of reaction between a chemical substance and receptor mole-cules. It is proposed that in the sucrose-sensitive units reaction occursbetween the anionic portion of saccharin sodium and sucrose-sensitivereceptor molecules (S-receptor molecules), while in the NaCl-sensitive unitsNa ions combine with Na+-sensitive receptor molecules.

Saccharin sodium presents a characteristic concentration-responsemagnitude relationship in units predominantly sensitive to sucrose, theresponse first occurring at 0 0003 M, reaching a maximum magnitude at

326


about 0-01-003 M and declining in magnitude with a further increase inconcentration. This is taken to indicate that the reaction between sac-charin molecules and S-receptor molecules at the receptor membrane showsan optimal concentration at about 0 01-0-03 M. Such a type ofreaction maybe considered to occur by the mechanism known as the non-competitiveauto-inhibition (Ariens, 1964). According to this, saccharin molecules havean affinity not only to the S-receptor sites, on which they induce propereffect but also to the receptor sites S', on which the effect originally inducedmay be depressed. At low saccharin concentration only the reaction withS-receptor sites occurs because of a greater affinity of saccharin with them,while with increasing saccharin concentration the reaction with S'-receptor sites commences and it begins to inhibit the original reaction.This would also explain the off-response observed when concentrated sac-charin sodium solution applied to the tongue was rinsed with water.

According to the results of preference experiments on rats by Beebe-Center,Black, Hoffman & Wade (1948) the preference threshold for saccharin sodium isabout 0-01 % (0-0004 M) and the maximally preferred concentration 0.5% (0.02 M).The results of the present experiments indicate that chorda tympani fibres of ratspredominantly sensitive to sucrose show a threshold concentration of 0-0003-0-001 Mfor saccharin sodium and a greatest magnitude of response at about 0-03 M, thusindicating similarity of the concentration-response magnitude relationship forsaccharin sodium in sucrose-sensitive units to the preference curve for this substance.In the hamster also the preference threshold for saccharin sodium is about 0-0005 Mand the maximally preferred concentration 0-02 M (Carpenter, 1956). Therefore, thepreference curve for saccharin sodium in the hamster is quite similar to the con-centration-response magnitude relationship in sucrose-sensitive units of hamsters.In addition, rats and hamsters show the preference threshold for sucrose at about0-015 M and the maximum preference at about 0-2 M (Richter & Campbell, 1940;Carpenter, 1956). In the present experiments the threshold concentration of sucrosefor the sucrose-sensitive unit was found to be about 0-01-0-03 M and its maximumresponse magnitude was found, in many cases, at 0-1-1 M. The concentration-response magnitude relationship for saccharin sodium agrees well with that forsucrose, if the former is shifted toward high concentration by 1-1-5 log units. Thisis in good agreement with the behavioural evidence that the saccharin sodium con-centration, which elicits a similar amount of intake in rats and hamsters to that ofsucrose, is 1-5 log units below the sucrose concentration (Beebe-Center et al. 1948;Carpenter, 1956). Such similarities of the concentration-response relationship forsucrose and saccharin sodium to the preference curve for these substances maysupport a concept that gustatory information for sucrose and saccharin sodium(sweet taste) is mediated mainly by impulse discharges in units predominantlysensitive to sucrose.On the other hand, the chorda tympani fibre responding exclusively to NaCl or

more sensitive to NaCl than to sucrose shows a very small magnitude of response to0-02 M saccharin sodium, where the sucrose-sensitive fibre produces a greatest re-sponse magnitude, and in such a unit the concentration-response magnitude re-lationship for saccharin sodium is similar in shape to that obtained for NaCl, thoughthe magnitude in the former is slightly smaller than that in the latter. Units of thiscategory are considered to mediate principally gustatory information for Na ions.

327

H. OGAWA, M. SATO AND S. YAMASHITAThere are, in addition, units of another category which is distinctively different fromthe above two because of their high sensitivity to HC1, quinine and sometimes toNaCl. These units were found to respond to saccharin sodium solution above 0-1 M.They may be assumed to be responsible principally for information for HCl andquinine.

The results of the present experiments bear some general significance forthe understanding of neural mechanism of gustatory sensations--Since thefinding that individual single units of the gustatory nerve respond to avariety of chemicals rather than those representing one of the four basicgustatory stimuli (Pfaffmann, 1941, 1955), specificity of taste units inmediating information of a particular quality of taste has been neglected,while an attempt to categorize taste stimuli into similarity classes by cal-culating correlations of the amount of neural responses across manyprimary neurones has gained success (Erickson, 1963; Erickson et al. 1965).However, Ogawa et al. (1968) and this present paper have indicated theexistence of several relatively distinct categories of units, each respondingparticularly well to one or a combination of gustatory stimuli. In addition,preference curves for sucrose and saccharin sodium are best explained onthe assumption that units predominantly sensitive to sucrose mediateprimarily information for sucrose and saccharin sodium and those elicitingimpulses particularly well in response to NaCl transmit information forNa ions. However, since there are only a few chorda tympani fibres in ratsand hamsters which are considered to respond only to one kind ofgustatorystimulus, specificity of units is not absolute. Therefore, discrimination ofquality of taste appears to depend on two complementary mechanisms,one being the relative specificity of taste units to a certain kind of stimulusand the other being the statistical difference of the neural response patternacross many neurones.To describe quantitatively neural information for saccharin sodium and

other chemicals, equations were derived in the present paper. Althoughthey are empirical and based on the impulse numbers, they indicate thatthe qualities of taste for a given chemical may be coded in terms of thosefor the four basic gustatory qualities. Consequently it would be possibleto extend this way of approach further to analyse gustatory responses tovarious other chemicals.

The authors are grateful to Mr T. Kiyohara for his help in some of the experiments.This research was supported by the U.S. Air Force Office of Scientific Researchthrough U.S. Army Research and Development Group (Far East) under Grants No.DA-CRD-AG-592-544-62-G30 and No. DA-CRD-AFE-S92-544-68-G121 and Con-tract No. DAJB17-69-C-0073.

328

GUSTATORY RESPONSE TO SACCHARIN 329

REFERENCES

ARIENS, E. J. (1964). Molecular Pharmacology, vol. I, p. 293. New York and London:Academic Press.

BEEBE-CENTER, J. G., BLACK, P., HOFFMAN, A. C. & WADE, M. (1948). Relativeper diem consumption as a measure of preference in the rat. J. comp. physiol.P8ychol. 41, 239-251.

CARPENTER, J. A. (1956). Species differences in taste preferences. J. comp. phy8iol.P8ychol. 49, 139-144.

COHEN, M. J., HAGIWARA, S. & ZOTTERMAN, Y. (1955). The response spectrum oftaste fibres in the cat: a single fibre analysis. Acta phy8iol. 8cand. 33, 316-332.

ERICKSON, R. P. (1963). Sensory neural patterns and gustation. In Olfaction andTaste, ed. ZOTTERMAN, Y. Oxford: Pergamon Press.

ERICKSON, R. P., DOETSCE, G. S. & MARSHALL, D. A. (1965). The gustatory neuralresponse function. J. gen. Phy8iol. 49, 247-263.

FISHER, G. L., PFAFFMANN, C. & BROWN, E. (1965). Dulcin and saccharin taste insquirrel monkeys, rats and men. Science, N.Y. 15, 506-507.

FISHMAN, I. Y. (1957). Single fiber gustatory impulses in rat and hamster. J. cell.comp. Phy8iol. 49, 319-334.

NAGAKI, J., YAMASHITA, S. & SATO, M. (1964). Neural response of cat to taste stimuliof varying temperatures. Jap. J. Physiol. 14, 67-89.

OGAWA, H., SATO, M. & YAMASHITA, S. (1968). Multiple sensitivity of chorda tym-pani fibres of the rat and hamster to gustatory and thermal stimuli. J. Phyaiol.199, 223-240.

PFAFFMANN, C. (1941). Gustatory afferent impulses. J. cell. comp. Phyaiol. 17,243-258.

PFAFFMANN, C. (1955). Gustatory nerve impulses in rat, cat and rabbit. J. Neuro-physiol. 18, 429-440.

RICHTER, C. P. & CAMPBELL, K. H. (1940). Taste thresholds and taste preferencesof rats for five common sugars. J. Nutr. 20, 31-46.

SATO, M., YAMASHITA, S. & OGAWA, H. (1969). Afferent specificity in taste. InOlfaction and Taate, vol. III, ed. PFAFFMAN, C. New York: Rockefeller UniversityPress. (In the Press.)

0-01m saccharin buffered with tris(hydroxymethyl)aminometh

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