Physiology and Behavior. Vol. 13, pp. 245-250. Brain Research Publications Inc., 1974. Printed in the U.S.A.

Chloroform Responses of the Chorda Tympani Nerve in the Rat

T A K A S H I Y A M A M O T O A N D Y O J I R O K A W A M U R A

Department o f Oral Physiology, Dental School, Osaka University, 32 Joancho, Kitaku, Osaka, Japan

(Rece ived 3 D e c e m b e r 1973)

YAMAMOTO, T. AND Y. KAWAMURA. Chloroform responses of the chorda tympani nerve in the rat. PHYSIOL. BEHAV. 13(2) 245-250 , 1974. - Electrophysiological analysis of the chorda tympani nerve response to saturated chloroform solution was performed in the rat, and the results were compared to those of the sucrose response. The chloroform response was characterized by a transient response, and it lacked an off-type response to the water rinse of the tongue. Treatment of the tongue with 0.1 M anionic detergent and 5 × 10 -4 M HgC12 produced the same effects on the chloroform and sucrose responses. However, the effects of some metallic ions on response to chloroform were different from those on the sucrose response. That is, 0.1 M CaCI~ suppressed the chloroform response and the off-type response, but 0.001 M CuCl~ suppressed only the sucrose response. Additive effect of sucrose and chloroform was rather smaller than the summation expected by doubling of the responses of sucrose and chloroform. The fibers which showed the off-type discharges also always responded to chloroform. These results suggest that sucrose and chloroform combine to the different loci within the same taste receptor macromolecule.

Chloroform Sucrose Chorda tympani nerve responses

C H L O R O F O R M general ly tas tes sweet to h u m a n s . Schallen- berger and Acree [7] d e m o n s t r a t e d in the i r molecu la r t h e o r y of sweet tas te t h a t m a n y c o m p o u n d s possess a c o m m o n molecu la r fea ture of a b i f u n c t i o n a l g roup cons is t ing of an acidic and a basic m o i e t y separa ted by a d i s tance of 2.5 to 4 A, and t hey also s ta ted t ha t c h l o r o f o r m was no t the excep t ion . Meanwhi le , Kur ihara [3] r epo r t ed t h a t gym- nemic acid A1 did no t suppress the sweet t as te of chloro- fo rm, a l t h o u g h it suppressed the sweet tas te of sugars, cyc lamate , D-amino acids, be ry l l i um chlor ide and lead ace ta te . This fact suggests t ha t r e cep to r m e c h a n i s m s in the c h l o r o f o r m response are d i f fe ren t f rom those in o t h e r sweet tas te subs tances .

To evaluate the tas te r ecep to r m e c h a n i s m s of the c h l o r o f o r m response, in the p resen t s tudy , an electro- physiological analysis of the responses of the chorda t y m p a n i nerve was p e r f o r m e d in the rat , and the resul ts were c o m p a r e d wi th those of the sucrose response.

METHOD

Thir ty-f ive Wistar a lb ino rats ( 1 8 2 - 2 3 6 g) were used. The an imal was deeply anes the t i zed by i n t r ape r i t onea l i n j ec t ion o f a m i x t u r e of N e m b u t a l (4 m g / 1 0 0 g) and u r e t h a n e (15 m g / 1 0 0 g) and was f ixed on a tab le in a side pos i t ion . The t r achea was cannu la t ed , and the masse te r muscle and m a n d i b u l a r ramus were t a k e n ou t and the cho rda t y m p a n i nerve was exposed u n d e r obse rva t ion wi th

a s t e r e o s c o p i c microscope . The nerve was careful ly separa ted f rom the su r round ing connec t ive t issues and was placed on a p l a t i n u m wire e lec t rode (100 u in dia.). The ind i f f e ren t e lec t rode was p laced on ad jacen t tissues. The e lec t rodes were c o n n e c t e d to a conven t i ona l ampl i f ie r (60 dB at the m a x i m u m ampl i f i ca t ion ) and the s u m m a t e d response of the nerve was r ecorded w i th an e lec t ron ic s u m m a t o r ( t ime c o n s t a n t : 0.5 sec) on an ink-wr i t ing re- corder. The response of a func t iona l ly single f iber to tas te s t imul i was r eco rded by a ca thode - ray osci l loscope and a record ing camera .

Reagent grade NaC1, CaC12, ZnC12, CuC12, HgC12, tar- tar ic acid, sucrose, ch lo ro fo rm , and J. P. quinine-HC1 were used as the tes t chemicals . Technica l grade ( > 9 7 % ) s o d i u m laury lsu l fa te (EMAL O: Kao-Atlas Co. Ltd . ) was used as a de tergent . All chemicals were dissolved in dist i l led wa te r (specif ic conduc t i v i t y < 5 × 10 -7 m h o / c m ) . As c h l o r o f o r m is a lmos t inso luble in water , a s a tu ra t ed c h l o r o f o r m solu- t ion was used. To make the sa tu ra t ed c h l o r o f o r m so lu t ion , the m i x t u r e of c h l o r o f o r m and wate r in a flask was s h a k e n v io len t ly by h a n d for a b o u t 1 min, and a f te r s t and ing for 2 m in the s u p e r n a t a n t was col lec ted.

Each chemica l so lu t ion of 15 ml was appl ied in 6 sec on the t ongue surface t h r o u g h a b u r e t t e f ixed jus t above the cen te r of the tongue . Af te r s t imula t ion , 3.5 ml of dist i l led wate r was del ivered by gravi ty f low on the t ongue by a n o t h e r bu re t t e , t h e n tap wate r was fo l lowed f rom a sys tem of an overhead funne l and a s topcock . The tas te so lu t ions and r insing wate r were kep t at 2 7 - 3 0 ° C.

245

246 Y A M A M O T O AND K A W A M U R A

RESULTS

The Sumrnated Response o f the Whole Chorda Tympani Nerve

The general p a t t e r n of a c h l o r o f o r m response was obvious ly d i f fe rent f rom tha t of a sucrose response. As s h o w n in Fig. 1, the pa t t e r n of the sucrose response was charac te r ized by a sus ta ined neura l act ivi ty and a s t rong burs t in response to the water rinse. On the o the r hand , the pa t t e rn of the c h l o r o f o r m response was charac te r ized by an init ial t rans ien t response wi th a small or w i t h o u t sus ta ined neura l act ivi ty (s teady s ta te response) , and the of f - type response was no t induced at the wate r rinse.

The previous t r e a t m e n t of the tongue surface wi th 0.1 M sod ium lau ry l su lpha te for 60 sec suppressed the successive acid and qu in ine responses, while this t r e a t m e n t did no t affect the responsiveness to NaC1, sucrose and to chloro- fo rm (Fig. 2). However , as s h o w n in Fig. 3, the previous t r e a t m e n t wi th 5 × 10 -4 M HgC12 for 60 sec suppressed b o t h sucrose and c h l o r o f o r m responses, bu t responses to the o the r 3 taste subs tances (NaC1, ta r ta r ic acid and quinine-HC1) were no t s ignif icant ly affected. Af te r each app l ica t ion o f the de te rgen t and HgC12, the tongue was r insed briefly unt i l the neura l act ivi ty to the de te rgen t and HgC12 r e t u r n e d to the b a c k g r o u n d activi ty.

Contro l

111 l I l l l l l l l l | I l l l I I | l l l I l l l l l | l l l [ l l l l l l l l l ~ l l I l l l

Sat, ~ ~

1M

t FIG. 1. Typical summated responses of the whole chorda tympani nerve to saturated chloroform and 1 M sucrose solutions. The chlo- roform response is characterized by a transient response without off-type discharges as compared to the sucrose response. Time in sec.

£1"cer Treatment ilill Ill,lil~'l, II Illl l l l l l ' l l l , l l l l , l l l l

O.OSW~ O.01N~

t t FIG. 2. Effects of 0.1 M anionic detergent applied to the tongue surface for 60 sec on the whole chorda tympani nerve responses to 4 taste stimuli and chloro- form solution. Note that the sucrose and the chloroform responses are not

affected. Time in sec.

CHLOROFORM RESPONSE 247

Control

t'""""t"'"""i'""'"'i"'

1M 8 u ~

Sat. ¢Jhlo~

O,1R~C1

0.05M T.£otd

0.01M L t n l ~ ~ Quinine

t

FIG. 3. Effects of 5 × 10 -4 M HgC12 60 sec on the whole chorda tympani and chloroform solution. Note that

form responses are strongly

The previous treatment of some metallic salts for 40 sec affected differently the sucrose response and the chloro- form response. Before application of the taste stimuli, the tongue surface was rinsed briefly until the neural activity to the metallic salt returned to the background activity. Re- suits obtained in 5 rats were summarized in Fig. 4. The values are presented by means +S.D. of 5 rats. The off-type response (striped bar) was suppressed to 15 +10% and the chloroform response (open bar) to 62 +4%, while the sucrose response (solid bar) was almost non-affected (95 -+5%) by previous application of 0.1 M CaC12. The sucrose, the off-type and the chloroform responses were suppressed to 68 +7%, 50 -+8% and 89 -+6%, respectively by 0.01 M ZnC12. The sucrose response was suppressed to 55 -+6%, while the off-type and the chloroform responses were not affected (95 -+7% and 102 +3%, respectively) by 0.001 M CuC12. These results suggest the existence of the different stimulating mechanisms of sucrose and chloroform in the receptor site of the tongue.

The neural responses to the mixture consisting of the sucrose and the chloroform solutions were compared to

after Treatment

t

applied to the tongue surface for nerve responses to 4 taste stimuli only the sucrose and the chloro- suppressed. Time in sec.

those to each of the individual stimulus to examine further the difference of the stimulating mechanisms between sucrose and chloroform. A typical example out of 4 cases is shown in Fig. 5. In the figure, a solid circle indicates the sucrose response at various concentrations, and an open circle shows the response to the mixture consisting of vari- ous concentrations of sucrose and the half-saturated chloro- form. The response magnitude to each solution was measured separately at the initial stage (Fig. 5-A) and at the steady stage at 10 seconds after the onset of stimulation (Fig. 5-B). The initial response to the mixed solution was always larger than that to the sucrose contained in that mixture, while the steady response to the mixed solution containing above 1/4 M sucrose was smaller than that to the individual sucrose contained in the mixture. To evaluate this diminution of the steady response quantitatively, the response magnitudes of the half-saturated chloroform solu- tion, 1 M sucrose and the mixture consisting of the half- saturated chloroform solution and 1 M sucrose were measured respectively and compared each other. When the steady state response to 1 M sucrose was counted as 100%,

248 YAMAMOTO AND KAWAMURA

1M 8uo1"oBe ~"~ ~ t , ~ l o x - o f o ~

i

10- ~D

¢-

° 8 Q. O3

= 6 o

(1) 4

= 2 t~

0.1M CaC12

+

÷

0.01M ZnC12

I I I I

/,/ //~ /F // // // // // // // // //

// // // // //

0.001M CuC12

FIG. 4. Effects of Ca ~, Zn ~ and Cu ++ on sucrose, off-type and chloroform responses. The sucrose response (solid bar) is almost not affected by 0.1 M CaC12, but suppressed by 0.01 M ZnC12 and 0.001 M CuC12 . The off-type response (striped bar) and the chloroform response (open bar) are not affected by CuC12 , but suppressed by

ZnCI~ and CaCI 2 . The control responses for each response are counted as 100%.

B Steady Response / I n i t i a l Response

o 40 2 o =

~- 30 15' o

X

½ ½ Conc. of Sucrose (i) Cone. of Sucrose (M)

FIG. 5. A typical concentration-response relationship for the sucrose solution, and the mixture of sucrose and chloroform. Magni- tude of response is measured separately at initial peak phase (A) and at steady phase (10 sec after onset of stimulation) (B). Solid circles represent the responses to the sucrose solution. Open circles indicate the responses to the mixture of various concentrations of sucrose solution and half-saturated chloroform solution. Horizontal bars in the graphs show the response magnitude of half-saturated chloro- form solution. Note that the response to the mixture (above 1/4 M

sucrose) is smaller than that to the sucrose solution in Graph B.

the s teady state response to the half-saturated ch lo ro form solut ion was 31 +11% (N = 4, mean +S.D.) and that to the mixture was 67 +11% (N = 4, mean +S.D.). These differ- ences were significant (t-test , p<0 .01) .

A Single Fiber Analysis of the Chorda Tympani Nerve

A single fiber analysis o f the taste response to chloro- form was per formed. A his togram summariz ing the fre- quency of response (number of impulses for the first one second) to the convent iona l 4 taste st imuli and to the saturated ch lo ro fo rm solut ion in the 16 d i f ferent fibers is shown in Fig. 6. In general, the fibers sensitive to sucrose also r e sponded to ch lo ro form, and the sucrose-insensit ive fibers did no t respond to ch loroform. Except ional ly , in some preparat ions (e.g. P in the figure), the sucrose- insens i t ive fibers were found which did respond to ch loroform.

Three d i f ferent f iber types were classified in the special reference to the responsiveness to sucrose and ch lo ro fo rm (Fig. 7). The actual recordings of these fibers in the figure are no t included in 16 fibers shown in Fig. 6. When the mode of response of these 3 fibers to 4 taste stimuli was de te rmined by the fol lowing cri ter ion; that is, when a

C H L O R O F O R M RESPONSE 249

'01 )o

20

lO

o

C h l o r _ o f o r m _

r,,n 4-,, 20

2",

Go 3 ° 1 II Oo.m_J n

°lj ,3o

20

10

0 SNAQC

N

8 N A QC

A

r 1. I!l I i _I!|1 ! IIq~!l~il?llllJlffllll~ll,,,j

I111111 I~ I ~ l l l l II I~ I I nllllll~ I Iltlll i l

B

' L l IN S u c r o s e i _ . j i i . . . . . . i l a - - _ ~ I - - - - II I 2

S I Ch

0 P C

Si t Ch)

SN A Q C SN A Q C

FIG. 6. A histogram summarizing the frequency of the chorda tympani nerve impulses to the 4 taste stimuli and to the saturated chloroform solution. Not all the sucrose sensitive fibers are sensitive to the saturated chloroform solution as shown in P. S: 1 M sucrose, N: 0.1 M NaCI, A: 0.05 M tartaric acid, Q: 0.01 M quinine-HC1 and

C: saturated chloroform solution.

response rate of the fiber during the first 5 sec of stimula- t ion increased over the spontaneous firing rate of this fiber, this f iber was classified as the posit ive fiber responding to this stimulus, then the fibers in Fig. 7-A, B and C were classified into sucrose-salt fiber, salt-acid fiber and sucrose- salt-acid fiber, respectively. The fiber in Fig. 7-A responded well to sucrose and ch loroform and also showed an obvious off- type discharge in response to the water rinse after sucrose s t imulat ion. This type of response was f requent ly observed. The fiber in Fig. 7-B did no t respond to sucrose, but obviously responded to the fol lowing water rinse, and this fiber also responded to ch loroform. This type of f iber characterist ically evoked a p redominan t discharge at the water rinse o f the tongue. The sucrose-insensitive fibers wi thou t this off - type discharge did no t show the chloro- form response, and such fibers as above-ment ioned sucrose and chloroform-insensi t ive fibers are c o m m o n l y observed as shown in Fig. 6. The third fiber in Fig. 7-C was sensitive to sucrose and insensitive to the water rinse. This fiber did no t evoke a s teady state ch lo ro fo rm response, but evoked only a transient response to the ch lo ro fo rm st imulat ion. This type of fiber was scarcely observed. These results suggest that the ch loroform response may be closely related with the generat ion o f the off - type response after sucrose adaptat ion.

J! i,mr I J,J!! J! l!! m , , v ~ , , r , ! , ,

!

'1 Std

FIG. 7. Three different fibers responsive to sucrose and chloroform. A fiber is sensitive to sucrose, water rinse and to chloroform. B fiber is not sensitive to sucrose, but sensitive to water rinse and to chloro- form. C fiber responds to sucrose without off-type response, and this fiber evokes only a transient response to the chloroform stimu- lation. The period of application of the chemicals was indicated by a

horizontal bar under the actual record.

DISCUSSION

Pre-application of an anionic detergent within a certain concent ra t ion under a mild condi t ion suppresses the succes- sive salt, acid and quinine responses, but this procedure does not affect the sucrose response [5] . The ch loroform response in the present exper iment was not affected by the anionic detergent , either. The sugar receptor in the taste receptor membrane is thought to be a sort of protein such as "sweet-sensit ive p ro te in" by Dastoli and Price [1] and Hiji e t al. [2] . The anionic detergent (0.1 M) with a specific aff ini ty to lipid may no t combine with the sugar receptor whose p roper ty is thought to be a protein. This may explain the fact that the sucrose response was unaf fec ted by the anionic detergent. On the contrary, 5 x 10-4M HgC12 suppressed only the sucrose response and did no t affect the responses to salt, acid and quinine solutions. The ch loroform response was also suppressed by this metall ic ion. The Hg ÷÷ ion is known to be a sulfhydryl reagent, hence it is possible that the suppression of the sucrose response by HgC12 may be brought about by some con- format iona l changes of sugar receptive macromolecule conta ining sul fhydryl groups. Nejad [4] observed that HgC12 produced a s trong irreversible inhibi tory effect on

250 YAMAMOTO AND K A W A M U R A

the NaCI response of the rat. But, when the low concentra- t ion of HgCI2 was used, only the sucrose response was inf luenced as shown in this study. Noma and Hiji [6] also observed that the low concent ra t ion of a sulfhydryl reagent (p-chloromercuric benzoate=PCMB) depressed only the sucrose response, while the higher concent ra t ion of PCMB depressed responses to all the 4 taste stimuli. This means that HgC12 as well as the anionic detergent , within an op t imum range of concentra t ion, might affect only a partic- ular kind of receptor site of the receptor membrane. The present results obta ined f rom the t rea tment of the tongue with HgC12 and the anionic detergent did not indicate any difference between the propert ies of the sucrose response and the ch loroform responses.

Meanwhile, the present s tudy also revealed that chloro- form and sucrose might have a different s t imulat ing act ion to the taste receptor. For example, the patterns of the whole nerve response to ch loroform and sucrose were different f rom each other, and the neural responses of both of these chemicals were different ly affected by the previous t rea tment of the tongue with CaC12 and CuC12. In addit ion, the nerve fibers responsive to sucrose were not always responsive to chloroform, and the steady response to the mixture of both chemicals was smaller than that to the

sucrose contained in the mixture. The mechanisms of the generat ion of the ch loroform

response and th~/t of the transient off- type response by the water rinse after sucrose adapta t ion seem to be similar, because both of these responses were not affected by CuC12 but suppressed by CaCI2 and ZnCI2 (Fig. 4), and the single fiber analysis revealed that the fibers showing a large and cont inuous ch loroform response were consistently respon- sive to the water rinse after sucrose application.

We proposed a concept recent ly that the off- type response induced after sucrose s t imulat ion is a water r e sponse originated from the sugar receptive macro- molecule, and the acceptor groups for water and sucrose exist separately within the same macromolecule ([8] manuscript in preparation). Fol lowing this idea, ch loroform may st imulate the water-sensitive-groups in the macro- m o l e c u l e concerning the generat ion of the off- type response, and may not st imulate the sugar-sensitive-groups in the macromolecule which have been proposed to be blocked compet i t ive ly by cupric ions [9 ,10] .

The present s tudy suggests that sucrose and ch loroform might combine to the different loci within the same macro- molecule. This may be a background mechanism of the insensitiveness of ch loroform to gymnemic acid A1 [3].

REFERENCES

1. Dastoli, F. R. and S. Price. Sweet-sensitive protein from bovine taste buds: isolation and assay. Science 154: 905-907, 1966.

2. Hiji, Y., N. Kobayashi and M. Sato. Sweet-sensitive protein from the rat tongue: its interaction with various sugars. Comp. Biochem. PhysioL 39B: 367-375, 1971.

3. Kurihara, Y. Antisweet activity of gymnemic acid A~ and its derivatives. Life Sci. 8: 537-543, 1969.

4. Nejad, M. S. Factors involved in the mechanism of stimulation of gustatory receptors and bare nerve endings of the tongue of the rat. Diss. Abstr. 22: 2855-2856, 1961.

5. Nitta, T., T. Yamamoto and Y. Kawamura. Effect of deter- gents on taste reception. J. physiol. Soc. Jap. 35: 576-583, 1973.

6. Noma, A. and Y. Hiji. Effects of chemical modifiers on taste responses in the rat chorda tympani. Jap. J. Physiol. 22: 393-402, 1972.

7. Schallenberger, R. S. and T. E. Acree. Molecular theory of sweet taste. Nature 216: 480-482, 1967.

8. Yamamoto, T. and Y. Kawamura. Studies on a water "Rinse Effect" after sucrose application to the tongue of the rat. J. physiol. Soc. Jap. 33: 294-302, 1971.

9. Yamamoto, T. and Y. Kawamura. Inhibitory effect of cupric and zinc ions on sweet taste response in the rat. J. Osaka Univ. Dent. Sch. 11: 99-104, 1971.

10. Yamamoto, T. and Y. Kawamura. A neurophysiological study on the taste of cupric ions. Jap. J. Physiol. 21: 359-374, 1971.