Amiloride Inhibits Taste Nerve Responses to NaC1 and KC1 in Sprague-Dawley and Fischer 344 Rats

Transcription

1 Physilgy & Behavir, Vl. 60, N. 2, , 1996 Cpyright 1996 Elsevier Science Inc. Printed in the US. ll rights reserved /96 $ ELSEVIER PII SO (96) milride Inhibits Taste Nerve Respnses t NaC1 and KC1 in Sprague-Dawley and Fischer 344 Rats MICHELLE M. MINER, SYMWONG E. HMMCK, ROBERT F. LUNDY, JR. ND ROBERT J. CONTRERS 1 Flrida State University, Department f Psychlgy, Tallahassee, FL US Received 8 ugust 1995 MINER, M. M., S. E. HMMCK, R. F. LUNDY, JR. ND R. J. CONTRERS. milride inhibits taste nerve respnses t NaCI and KCI in Sprague-Dawley and Fischer 344 rats. PHYSIOL BEHV 60(2) , In a tw-bttle test, Sprague-Dawley rats preferentially cnsume a greater amunt f hyptnic and istnic NaC1 slutins relative t water, whereas inbred Fischer 344 (F344) rats fail t prefer NaC1 slutins at any cncentratin relative t water. T determine whether taste cntributes t this strain difference, we measured the integrated neural respnses f the chrda tympani nerve t a cncentratin range f NaC1 and KCI slutins. The amilride-sensitive cmpnent f the taste nerve respnse was assessed by adding amilride during salt stimulatin in Experiment 1, and by pretreating the taste receptrs with amilride prir t salt stimulatin in Experiment 2. dding amilride t NaC1 during sustained neural activity suppressed chrda tympani nerve respnses mre than pretreating the tngue with amilride. dding amilride during salt stimulatin als partially suppressed chrda tympani neurn respnses t KCI, a presumed cntrl stimulus. The neural respnses f the chrda tympani nerve t NaC1 and KC1 were similar fr salt-aviding F344 and salt-preferring Sprague-Dawley rats. Hwever, amilride pretreatment suppressed the taste nerve respnses t NaC1 significantly less in F344 rats than in Sprague-Dawley rats. The strain difference in the amilride-sensitive cmpnent f the taste respnse may cntribute t the difference in NaCI preference. Salt preference Strain difference Receptr mechanisms Taste electrphysilgy Chrda tympani Nerve LTHOUGH sdium is an essential nutrient that must be cnsumed, there are large differences in NaC1 intake amng mammals. Sme humans, rats, and mice presumably like the taste f NaC1 and will cnsume it in large quantities, whereas thers d nt (15). Fr example, when given a tw-bttle intake test between water and NaCI slutin, many rat strains, as the utbred Sprague-Dawley, will preferentially cnsume a greater amunt f hyptnic and istnic NaC1 slutins relative t water (9,13). In cntrast t the Sprague-Dawley strain, the inbred Fischer 344 (F344) strain fails t prefer NaCI slutins at any cncentratin relative t water (34). This strain difference in intake seems t be unique t NaCI, as F344 rats' preference r aversin fr sweet, sur, and bitter tasting slutins were similar t that f ther strains. dditinally, F344 rats' respnse t sdium depletin by increasing hypertnic saline intake is mdest at best cmpared t the rbust salt appetite exhibited by ther strains (35). The difference in NaC1 intake between F344 and Wistar rats appears t be unrelated t differences in Na + excretin. Taste is the primary sensry system that functins in the detectin, recgnitin, and ingestin f NaCI. The chrda tympani nerve, which innervates the taste receptrs n the anterir tw-thirds f the tngue, is highly respnsive t the mnchlride salts, especially NaC1 (3,22). fter bilateral transectin f chrda tympaui nerves, rats trained t avid the taste f NaC1 exhibited a 50-fld increase in detectin threshlds (46) and impaired ability t distinguish NaC1 frm KC1 (45). Transectin f the glsspharyngeal nerve, which innervates the taste receptrs n the psterir ne-third f the tngue, did nt alter NaC1 and KCI discriminatin. Several studies have shwn that sdium salts stimulate taste receptrs by Na + influx thrugh in channels n the apical plasma membrane f taste receptr cells (1,2,7,17,26,36). milride hydrchlride, an epithelial Na + channel blcker, reduced the inward current prduced by sdium ins (26,36), and partially suppressed the integrated respnses f the salt-sensitive chrda tympani nerve t NaC1 in rats (7,16,26), mice (23,39), hamsters (28), dgs (36,37), and rhesus mnkeys (27). Insfar as amilride treatment never cmpletely suppressed taste nerve respnses t NaCI, it appears that sdium taste prcessing invlves bth amilride-sensitive and amilride-insensitive transductin pathways (16,21). The results frm behaviral studies are generally cnsistent t T whm requests fr reprints shuld be addressed. 507

2 508 MINER ET L. with findings frm physilgical studies. In human behaviral studies, amilride suppressed NaC1 taste intensity (40,42) and the saltiness f NaCI in sme cases (20,33,47). In rat behaviral studies, amilride treatment mdified intake and licking rate t NaC1 slutin, suggesting perhaps that amilride altered the rats' taste sensatin f NaC1 intensity and/r quality (5,11,14,30,32). Taste appears t underlie the strain difference in NaCI intake between F344 and salt-preferring Wistar rats. s demnstrated with taste reactivity test in rats, micrinfusin f taste slutins directly int the muth elicit steretypical patterns f muth and bdy mvements. These taste-elicited reflexes are divisible int psitive ingestive and negative aversive respnses that cincide with the intensity and quality f the taste stimulus (25). Using taste reactivity, F344 rats made taste-elicited aversive respnses t all NaCI cncentratins, whereas salt-preferring Wistar rats made n aversive respnses and many ingestive respnses (24). Bilateral transectin f chrda tympani nerves in F344 rats reversed their NaCI aversin evident in a tw-bttle intake test s that they preferred hyptnic and istnic NaC1 slutins like ther rat strains (44). The mean relative respnses f the chrda tympani nerve t a brad range f NaC1 cncentratins was greater in F344 than in Wistar rats; a larger amilride-sensitive cmpnent appears t be respnsible fr the greater NaCI respnse in F344 rats (6). The Sprague-Dawley rat has been the dminant experimental mdel fr investigatins dealing with salt intake behavir, salt appetite, and peripheral encding mechanisms f salt taste. With few ntewrthy exceptins (6,34,35), the Wistar rat is infrequently the subject f chice in studies f salt taste and salt appetite. The F344 rat is the predminant experimental mdel fr cancer and aging research. There may be nly ne electrphysilgical study f salt taste respnses in F344 rats (6). The uniqueness f F344 rats' behaviral respnse t NaC1 makes an electrphysilgical cmparisn t the well-knwn Sprague-Dawley strain f special interest. We therefre cmpared the integrated respnses f the chrda tympani nerve in F344 rats with the mre widely studied Sprague-Dawley rats. The present study included a brad range f NaC1 and KCI cncentratins with and withut amilride. There are n prir studies f chrda tympani nerve respnses t KCI in F344 rats. In the present study, amilride suppressin f chrda tympani nerve respnses t salt stimulatin invlved tw prcedures. First, we measured the magnitude f the respnse decrement that ccurred frm adding amilride t nging NaCI stimulatin. Secnd, we measured NaCI respnse amplitudes after pretreating the tngue with amilride and cmpared them t thse after water pretreatment. The frmer is a relatively new prcedure perhaps mre sensitive in detecting the amilride-sensitive cmpnent f the taste nerve respnse (28). The latter is the mre cmmn prcedure in studies f NaCI taste transductin. We used bth prcedures t maximize the pssibility f uncvering a strain difference in salt taste sensitivity. s far as we knw, this is the first time bth amilride suppressin prcedures have been cmpared in the same study. Subjects EXPERIMENT 1 METHOD Data fr 0.3, 1.0, and 10 ~M amilride were btained frm nine adult male Sprague-Dawley and nine F344 rats, with mean weights f 454 and 276 g, respectively. Data fr 100 I~M amilride were btained frm three adult Sprague-Dawley rats (tw female and ne male) with a mean weight f 436 g and fur adult F344 rats (three female and ne male) with a mean weight f 285 g. The F344 rats were typically 100 g smaller than Sprague-Dawley rats f similar age. ll animals came frm the Wilmingtn & Raleigh divisins f Charles River Labratries. nimals lived individually in standard wire hanging cages in a rm n a 12:12 light dark cycle with lights n at 0500 h. The animals had free access t Purina Rat Chw 5001 and distilled water. Neural Recrding Prcedure Surgeries began apprximately 4 h int the animals' light cycle. nesthesia cnsisted f urethane (1.5 g/kg bdy weight) given in tw IP injectins spaced 15 min apart. Supplements f 0.1 ml were given as needed t maintain a deep level f anesthesia. The animals received a trachetmy. heating pad maintained bdy temperature between 36 and 38 C thrughut recrding. nntraumatic head hlder secured the animal's head. The chrda tympani branch f the seventh cranial nerve was expsed using a mandibular apprach. The verlying auricultempral, mylhyid, and inferir alvelar nerves were transected and remved. The chrda tympani was then islated frm where it jins the lingual nerve t the bulla where it was cut. The peripheral end was desheathed and placed upn a nichrme wire recrding electrde. similar indifferent electrde was placed int nearby tissue. The nerve was cvered and bathed with mineral il t prevent drying. The tngue was secured and psitined with a length f suture and remained cvered with a cttn wad saked in deinized water thrughut surgery. Chrda tympani nerve respnses were differentially amplified 1000, full wave rectified, and integrated with a Grass 7P3b R-C integratr (time cnstant 0.2 s). Respnses were recrded n a Grass plygraph fr later quantificatin and stred n a Vetter vide cassette recrder. ll respnses were simultaneusly mnitred via a Tektrnix scillscpe and a Grass audi mnitr. Slutins Test slutins f NaCI, KC1, NH4CI, and amilride hydrchlride were made frm dubly distilled water and chemicals btained frm Sigma Chemical Cmpany. They were presented at rm temperature (22-24 C). Stimulus Prtcl Test slutins were delivered t the tngue by gravity flw frm 60-ml syringes at an apprximate rate f 3 ml/min. cncentratin series was always bracketed by a s delivery f 0.1 M NHaC1 t ensure the stability f the respnse ver time. t all ther times the tngue was cntinually bathed by a cnstant flw f distilled water. NaCl and KCI The first stimulus series determined the nrmal peak and tnic respnses f the chrda tympani nerve t an ascending series f NaCl and KCI cncentratins (0, 0.03, 0.1, 0.3, and 1.0 M). Each stimulus was presented fr 10 s. minimum 1-min distilled water rinse preceded each test stimulus (this was lnger fr strng cncentratins). T assess the specificity f amilfide hydrchlride inhibitin, KCI was used as stimulus. In rats, amilride is significantly less effective in inhibiting chrda tympani nerve respnses t KCI as cmpared t NaCI (7,16,29).

3 MILORIDE INHIBITS TSTE 509 I in 0,3 amilride -'-I O,03MNaCL FIG. 1. Representative integrated respnse f the whle chrda tympani nerve t NaCI stimulatin with and withut amilride. The bttm hrizn line represents the entire 30-s perid f stimulatin. The upper hrizntal line represents the middle 10-s segment when amilride was added. milride The secnd stimulus series examined the suppressive effects f 0.3, 1.0, 10, and 100 txm amilride n chrda tympani nerve respnses t an ascending series f NaCI and KC1 cncentratins (0, 0.03, 0.1, 0.3, 1.0 M). Test stimuli were presented fr 30 s fllwed by a 1-min distilled water rinse (lnger fr strng cncentratins). The 30-s stimulatin perid was divided int three cntinuus 10-s segments with the stimulus presented alne, mixed with the amilride, and again alne, respectively (see Fig, 1 fr an example). given animal was tested with nly ne amilride cncentratin. Respnse Quantificatin and nalysis The chrda tympani respnses t each salt slutin were standardized t the mean peak respnse t 0.1 M NH4C1 befre and after a cncentratin series. The peak respnse was measured as the maximum respnse amplitude ccurring within 2 s after stimulus nset, and the tnic respnse as the respnse amplitudes ccurring 5 and 10 s after stimulus nset. milride suppressin was scred as the rati in respnse magnitudes during amilride (secnd 10-s segment) relative t the average cntrl respnses befre and after amilride (first and third 10-s segments). The cntrl respnses were measured 2 s befre and 4 s after amilride and averaged. Measurement at these time pints allwed fr respnse stabilizatin and cntrlled fr pssible prgressive changes in the chrda tympani nerve respnse ver time. milride respnses were measured 5 and 10 s after amilride nset. milride suppressin was cmputed using the equatin: 100{amilride respnse/[(tnicl + Tnic2)/2]}. Suppressin scres culd nt be cmputed fr 0 M cncentratins f NaC1 and KCI because the tnic activity was either t small r lacking fr these salt cncentratins t bserve an amilride effect. Statistical analysis was dne using analysis f variance (NOV) tests with repeated measures n Strain, Tastant (NaC " 1 1 Phasic Respnse ~. 100" 100- ~ ~ 4 4 (J ~ F344 ~. 0.(]I....'I... i 1.i.I s Tnic Respnse ~ 10-s Tnic Respnse n 100, 80- ~, "6,.) 60- ~ SD F NeCI Cncentratin (M) KCl Cncentratin (M) FIG. 2. The mean phasic and tnic respnses t NaC1 and KCI relative t 0.1 M NH4CI in Fischer 344 and Sprague-Dawley rats.

4 510 MINER ET L. r KCI), milride Cncentratin, and Time f Measure variables. Pst hc analyses were perfrmed using univariate F and Wilk's lambda multivariate F-tests. Significant differences were reprted as p < In additin, paired samples t-tests were cnducted n salt cncentratin (NaCI vs. KC1) acrss amilride cncentratin. NaCl and KCl Respnses RESULTS Neural respnses were expressed as a percentage f the respnses t 0.1 M NHaCI, which were similar (F ; SD ) in magnitude between the tw strains. s can be seen in Fig. 2, the peak and 10-s tnic respnses f the chrda tympani nerve increased with NaC1 and KCI cncentratin (all p < ). There were n differences between F344 and Sprague-Dawley rats in either mean peak respnses t NaCI, F(I, 16) 1.406, p , r t KCI, F(1, 16) 0.157, p There were als n strain differences in the 10-s tnic respnses t NaC1, F(1, 15) 2.153, p 0.163, r t KC1, F(1, 16) 0.957, p Similar results were btained fr the 5-s tnic measure. NaCI Suppressin s can be seen in Fig. 3, the suppressin functins fr 0.3, 1.0, 10, and 100 p.m amilride were similar fr F344 and Sprague-Dawley rats, F(3, 16) 0.193, p 0.9. There was a significant main effect fr milride Cncentratin, F(3, 16) , p < , as suppressin increased with cncentratin with 100 IxM > 10 ~M > 1.0 IxM 0.3 IxM (all p < 0). Figure 3 als shws that amilride's effectiveness varied acrss NaCI cncentratin. Bth 0.3 ~M, F(1, 4) , p 0.001, and 1 I~M amilride, F(1, 4) , p 0.001, were effective nly fr the tw weakest NaC1 cncentratins. milride at cncentratins f 10 and 100 p,m suppressed chrda tympani respnses t all fur NaC1 cncentratins. Fr all fur amilride cncentratins, the amunt f suppressin tended t decline with increasing NaCI cncentratin, particularly fr 0.3 and 1.0 txm amilride. KCl Suppressin Cntrary t ur expectatin, amilride significantly suppressed KCI respnses. There was a significant main effect fr milride Cncentratin, F(3, 16) 7.648, p 0.002, as suppressin increased with cncentratin (100 ~,M 10 ~M and 100 IxM > 1.0 IxM 0.3 IxM; all p < 0.05). s can be seen in Fig. 4, the suppressin functins fr 0.3, 1.0, 10, and 100 IxM amilride were similar fr F344 and Sprague-Dawley rats, F(3, 16) 1.657, p Figure 4 shws that suppressin f KC1 respnses, althugh less than suppressin f NaCI respnses, als increased as amilride cncentratin increased. Figure 4 als shws that amilride's effectiveness varied acrss KCI cncentratin. Bth 0.3 ~M, F(1, 4) 9.342, p 0.002, and 1.0 ~M, F(1, 4) 2.797, p 0.086, amilride were 1 0,3 ~M mllrlde ~.M mllrlde 100" t- O -i U) SD F3~ t--.2 P l. -.i ' F344 O" 0" , , ,, p.m mllride ~.M milrlde _ a. a Z ~ _ F F~4 0., 0" ' , , , , , , , NaCl Cncentratin (M) NeCl Cncentratin (M) FIG. 3. The average percent suppressin f chrda tympani nerve respnses t fur cncentratins f NaC1 with 0.3, 1.0, 10, and 100 p.m amilride in Fischer 344 (F344) and Sprague-Dawley (SD) rats.

5 MILORIDE INHIBITS TSTE 511 effective in suppressing chrda tympani nerve respnses t nly 0.03 M KCI. This was nt the case fr 10 I~M, F(1, 4) 0.938, p 0.453, r 100 IcM, F(1, 4) 1.717, p 0.217, fr which suppressin was very similar acrss KCI cncentratins. milride cncentratins f 10 and 100 p~m suppressed chrda tympani respnses t all fur KC1 cncentratins. Fr all fur amilride cncentratins, the amunt f suppressin tended t decline with increasing KC1 cncentratin. NaCI and KCl Cmparisns Because there were n significant strain differences in amilride suppressin, the data frm bth strains were cmbined t cmpare NaC1 and KC1 suppressin. milride suppressin differed significantly by salt, F(7, 40) , p < Pst hc analysis (see Fig. 5) indicates that NaC1 and KCI respnses begin t diverge significantly nly in respnse t 10 and 100 IxM amilride. s shwn in Fig. 5, 10 ixm amilride suppressed 0.1 M, F(1, 40) 4.306, p 0.044, and 0.3 M, F(1, 40) 8.248, p 0.006, NaC1 t a greater extent than the same cncentratins f KC1. With 100 p~m amilride, there was even a greater divergence in suppressin amng 0.1 M, F(1, 40) , p 0.001, and 0.3 M, F(1, 40) 73.95, p < 0.001, salt cncentratins. There were n differences in suppressin between 0.03 M NaCI and KCI and between 1.0 M NaC1 and KC1 with 10 ~M r 100 p.m amilride (all p > 0.05). Respnse Recvery Frm milride Suppressin respnses were scred at fur times, 5 s apart. The first and secnd time samples ccurred during amilride applicatin. The last tw were taken during recvery f the respnse t the salt stimulus. This was dne t reveal any pssible differences in sensitivity acrss time between the tw strains. Strain x Time effect was nt fund fr either NaC1, F(3, 48) 1.809, p < 0.158, r KC1, F(3, 48) 0.828, p < EXPERIMENT 2 In Experiment 1 the integrated respnses f the whle chrda tympani nerve t a brad range f NaC1 and KC1 cncentratins were similar fr F344 and Sprague-Dawley rats. Furthermre, the degree f NaC1 taste suppressin with a brad range f amilride cncentratins was similar fr F344 and Sprague- Dawley rats. Unlike the results f sme prir studies (7,26), amilride als suppressed the neural respnses t KCI slutins. This was the first study t use amilride additin t examine a strain difference in salt taste sensitivity and assess the specificity f amilride blckade. We cnducted Experiment 2 t determine whether ur bservatins were prcedurally specific t the way f measuring amilride suppressin " 0,3 I~M milrlde 10 p.m milrlde 100" 100 " e" O D. O. -i fj SO F3~ -.2 P D. in -i U) 60 40' 20' SO Fa44 0' i , , , , , , 120' 1.0,~M mllrlde 120 " 100 ItM milrlde 100' 1 O0" 80'.-e e".2 t~ 60' SO F344 _,,1 (/1 SO " ~ F344 O" , , , , ,....., , " " " 10 KCI Cncentratin (M) KCl Cncentratin (M) FIG. 4. The average percent suppressin f chrda tympani nerve respnses t fur cncentratins f KC1 with 0.3, 1.0, 10, and 100 IxM amilride in Fischer 344 (F344) and Sprague-Dawley (SD) rats.

6 512 MINER ET L. METHOD Whle nerve recrdings were btained frm six adult F344 (mean 359 g) and six adult Sprague-Dawley (mean 661 g) rats in respnse t stimulatin with an ascending cncentratin series f NaC1 (0.06, 0.1, 0.3, and 1.0 M NaCI) and t 0.1 M NHnCI. Respnses t 0.1 M NHaCI befre and after each cncentratin series were used t assess the stability f neural recrding. The cmplete set f NaC1 cncentratins was tested using distilled water as the rinse slutin, a 1-min rinse preceding each taste stimulus. The same NaCI slutins were then tested with 100 IxM amilride hydrchlride as the rinse slutin (23). With a new stimulus delivery system (31), taste slutins and rinses were presented t the tngue at a precisely cntrlled flw rate f 25 ~l/s and temperature f 25 C. The data were stred n a vide cassette recrder and analyzed ff-line n a Pwer PC cmputer equipped with a GW Instrument 15-p,s data acquisitin bard and SuperScpe II data analysis sftware. The neural respnses were quantified by the area under the 20-s respnse curve using SuperScpe II. ll ther cnditins were identical t thse in Experiment 1. RESULTS S was shwn in Experiment 1, there were n differences in relative NaCI respnses after water adaptatin between F344 and Sprague-Dawley rats, F(1, 40) 0.308, p There were als n strain differences in relative NaC1 respnses after amilride adaptatin, F(1, 40) 1.491, p lthugh there were n strain differences fr either rinse cnditin, the neural respnses t 0.06 and 0.1 M NaCI tended t be smaller after the water rinse in F344 rats ( ; ) cmpared t Sprague-Dawley rats ( ; ). The ppsite was seen after amilride rinse as the respnses t 0.06 and 0.1 M NaC1 tended t be larger in F344 rats ( ; ) cmpared t the same respnses in Sprague-Dawley rats ( ; 0.49 _+ 0.06). These trends fr the tw weakest NaC1 cncentratins are evident by cmparing the NaC1 respnse functins fr F344 and Sprague-Dawley rats (see Fig. 6). s supprted by a main effect fr Rinse, amilride significantly suppressed the chrda tympani nerve respnses t NaCI in bth F344, F(1, 40) 5.973, p 0.02, and Sprague-Dawley rats, F(1, 40) , p < s revealed in pst hc tests, the surce f this suppressin was due t the respnses t 0.3 M (p0.03) and 1.0 M (p0.004) NaCI in F344 rats. In Sprague-Dawley rats, the surce f the amilride suppressin was due t 0.1 M (p 0.048, ne-tailed), 0.3 M (p < ), and 1.0 M (p ) NaC1. Given these NOV results and by cmparing the functins shwn in Fig. 6, amilride suppressin seemed t be mre significant fr Sprague-Dawley rats than 1 100" 0.3 p.m mllrlde IxM mllride 80- t- O. (/) 40- KCI r NaCI Kel 0" I I p.m milrlde p.m milride O0 " 80- e~.2 t P a. D. u~ 60- NaCI I _ ~ I~, r/) 20- ~ NaCI ~ Kcl 0- O-,1 1 Salt Cncentratin (M) Salt Cncentratin (M) FIG. 5. cmparisn f amilride suppressin between NaCI and KCI respnses. Because there were n strain differences, the data frm Fischer 344 and Sprague-Dawley rats were cmbined.

7 MILORIDE INHIBITS TSTE 513 C O O rr 3,0 ~ 2.5" 2.0' 1.5' 1.0' T Fischer 344 Rats.L Water Pretreatrnent rnitride Pretrealment ve Cmpnent DISCUSSION In tw separate experiments, we fund that the integrated respnses f the chrda tympani nerve t NaC1 were similar fr salt-aviding F344 and salt-preferring Sprague-Dawley rats. We did, hwever, find a small strain difference in the amilride-sensitive prtin in the chrda tympani respnse t NaC1 after amilride pretreatment, but nt after the additin f amilride during NaC1 stimulatin. milride partially suppressed chrda tympani neurn respnses t NaCI and unexpectedly as well t KC1, a presumed cntrl stimulus. -0.5,01 3.0" ~. ~ 1.0" _m " Sprague-Dawley Rats Water Pretreatment milride Pretreatment ve Cmpnent " ,.... ' 01 1 t " " " 10 NaCl Cncentratin FIG. 6. The average chrda tympani respnses t NaC1 relative t 0.1 M NH4C1 after pretreatment with distilled water r 100 IxM amilride in Fischer 344 (tp) and Sprague-Dawley (bttm) rats. The amilride-sensitive cmpnent f the nerve respnse was determined by subtracting the respnses after amilride pretreatment frm the respnses after water pretreatment. fr F344 rats, particularly fr the tw weakest NaCI cncentratins. Figure 7 presents a direct cmparisn f amilride suppressin f the fur NaC1 cncentratins between F344 and Sprague-Dawley rats. There was a significant main effect fr strain indicating that amilride suppressin was greater fr Sprague-Dawley rats, F(1, 40) 8.619, p The surce f this strain difference was due t 0.06 M (p 0) and 0.1 M (p0) NaCI, but nt fr 0.3 M (p0.511) r 1.0 M (p 0.976) NaC1. CL cn 50" 30" 10 0" -10 (M)!. SD - ~ F NaCl Cncentratin (M) FIG. 7. The average percent suppressin f chrda tympani nerve respnses t fur cncentratins f NaCI with 100 p.m amilride pretreatment in Fischer 344 (F344) and Sprague-Dawley (SD) rats. Strain Differences in NaCI Taste Preference We expected ur results t be similar t prir wrk (6) cmparing F344 rats with salt-preferring Wistar rats and finding a larger relative NaC1 respnse and greater amilride sensitivity in F344 rats. If salt taste mechanisms play a rle in salt preference, then ne may expect salt-preferring Wistar and Sprague- Dawley rats t be similar in relative neural respnse and amilride sensitivity and equally different frm F344 rats. Hwever, we classify Wistar and Sprague-Dawley as salt preferring nly frm their preference perfrmance in tw-bttle intake tests. In these lng-term tests, taste as well as many pstingestive factrs, cntributes t the preference behavir. s far as we knw, there are n studies directly cmparing Wistar and Sprague-Dawley either in lng-term r in brief expsure tests that minimize pstingestive factrs. The pssibility exists that Wistar rats may als differ frm Sprague-Dawley rats in their salt taste behavir. If this were the case, then ne may expect Wistar and Sprague- Dawley rats t differ as well in chrda tympani respnse and amilride sensitivity. In rat studies f taste and salt intake behavir, the Sprague- Dawley is the animal mdel mst ften used in the experimental literature. The salt-sensitive chrda tympani f Sprague-Dawley rats respnds strngly t a brad range f cncentratins. The relative NaC1 respnses fund in the present study are similar t thse reprted by us (12) and thers (7,21,26) in the experimental literature. There may be nly ne electrphysilgical study f salt taste respnses in F344 rats (6). The Wistar rat is als an uncmmn mdel fr electrphysilgical taste research. Because f its lng histry f use in taste electrphysilgy, the Sprague-Dawley may be a better mdel with which t cmpare chrda tympani respnses with ther strains like the F344. Despite sme differences in the anesthetic, stimulus prtcl and flw rate, integratr time cnstant, and nrmalizing NH4C1 cncentratin, the relative NaC1 respnses f F344 frm the present and earlier study (6) cmpare favrably. The neural respnses f F344 t strng NaC1 cncentratins are larger than the nrmalizing NH4C1 stimulus in bth the earlier and present study, particularly in Experiment 2. The NaCI respnses f the chrda tympani in Wistar rats were smaller than the nrmalizing NHaC1 stimulus in the previus study (6). Bernstein and her cwrkers (6) fund the threshld amilride cncentratin fr suppressin t be at least as lw as 1 ixm amilride in F344 cmpared t the mimimum f 5 IxM in Wistar. In the present study, 0.3 IxM amilride was equally effective in suppressing chrda tympani respnses t NaC1 in F344 and Sprague-Dawley. Thus, the amilride threshld fr F344 and Sprague-Dawley appears t be the same. Bernstein, et al. (6) fund greater NaC1 suppressin using a higher 500 IxM amilride cncentratin t blck all amilride-sensitive channels than the 100 IxM cncentratin used in the present study. Hwever, a higher amilride cncentratin shuld nt change the pattern f results fund in the present study.

8 514 MINER ET L. Hill and his assciates (30) cnducted an fmprtant study in rats suggesting that the amilride-sensitive prtin f the functinal taste respnse t NaCI mediates a taste assciated with sdium catins, whereas the residual amilride-insensitive prtin mediates a taste assciated with nnsdium catins. Cntrl rats trained t avid the taste f 0.5 M NaCI generalized their aversin nly t sdium salts specifically t 0.1 M NaCI and t 0.1 and 0.5 M sdium acetate. milride-treated rats, in cntrast, generalized their cnditined aversin f 0.5 M NaC1 t 0.1 and 0.5 M cncentratins f KC1, NH4CI, and ammnium acetate, but nt t sdium salts. Furthermre, rats prefer NaCl ver KCI and NI-I4CI as revealed in shrt-term intake (10) and in lick rate (8) tests in water-deprived rats. Frm these findings, the amilride-sensitive cmpnent seems necessary t distinguish between the preferred sdium salts frm the nnsdium salts. In Experiment 2, there was a small strain difference in the amilride-sensitive prtin f the chrda tympani nerve as assessed by the relative respnse amplitudes t NaCl after water and amilride adaptatin. In F344 rats, an amilride-sensitive cmpnent f the chrda tympani nerve respnse was evident during stimulatin with hypertnic 0.3 and 1.0 M NaC1, but nt during stimulatin with hyptnic 0.06 and 0.1 M NaC1. In Sprague-Dawley rats, an amilride-sensitive cmpnent was evident fr all f the NaCl cncentratins. With taste reactivity tests, Grill and Bernstein (24) demnstrated that F344, but nt Wistar, reacted aversively t hyptnic and istnic NaCl cncentratins. Sprague-Dawley rats reacted with psitive ingestive respnses up t 0.3 M NaC1, and aversively t higher NaC1 cncentratins (20). If the amilride-sensitive prtin f the chrda tympani nerve respnse underlies the sdium taste in rats, then frm Hill's wrk (30) we can infer that all NaCl cncentratins elicit the same sdium-driven taste but vary in intensity acrss the cncentratin spectrum in Sprague-Dawley rats. T F344 rats hypertnic saline may elicit the sdium taste, but hyptnic saline may elicit anther taste because f the absence f amilride sensitivity. The excessively high cncentratin f hypertnic saline may cntribute t the aversin f nrmally preferred sdium slutins in salt-preferring and salt-aviding rats. The unpalatable, perhaps nnsdium quality may cntribute t F344 rats' aversin fr hyptnic and istnic NaC1 slutins. lthugh this may be an intriguing explanatin, the strain difference in amilride sensitivity is small. The difference des nt appear t be large enugh t accunt cmpletely fr the striking behaviral difference between the tw strains. The difference in amilride sensitivity may cntribute t the NaCl aversin in F344, but a central change in the evaluatin f salt taste must als ccur. milride Suppressin f Whle Nerve Respnses dding amilride t NaCl during sustained neural activity suppressed chrda tympani nerve respnses mre than pretreating the tngue with amilride. Fr example, in Sprague-Dawley rats we fund that adding 100 plm amilride t the fur NaC1 cncentratins suppressed the sustained tnic respnses by an average f 65%, whereas pretreatment suppressed the respnses by nly 34%. In F344 rats, adding 100 p~m amilride t the stimulating NaC1 slutin suppressed NaCl respnses by 68%, whereas pretreatment suppressed the respnses by 18%. This is the first instance t cmpare the tw amilride prtcls within the same study. The present results are cnsistent with previus findings suggesting that amilride additin may be a mre sensitive methd f assessing suppressin than pretreatment (28). There are at least tw reasns why adding amilride t the stimulating NaCl slutin may be mre effective than pretreat- ment (28). The duratin f amilride applicatin, which is always lnger with pretreatment than with additin, may be ne key factr. In studies using pretreatment, amilride applicatin may be as lng as 5 min befre the nset f an ascending cncentratin series (7,26) cupled with shrter rinses f 1-min duratin befre each stimulus. When added t the stimulating NaC1 slutin, amilride was present fr nly 10 s. The reversal f amilride suppressin after prlnged pretreatment may take minutes (16). The reversal after amilride additin may take nly a few secnds (28), as was bserved in the present investigatin. Lengthy pretreatment may alter the taste receptr membrane in a way t reduce amilride suppressin. Fr example, amilride pretreatment may reduce the spntaneus firing frequencies f chrda tympani neurns (7), reflecting an alteratin in the resting membrane characteristics f the taste cells. The differential effect f amilride n s called "N" and "H" fibers may be a secnd reasn why amilride additin is mre effective than pretreatment. N and H fibers cnstitute in equal numbers the majr fiber grups within the chrda tympani nerve (22). Bth fibers respnd well t NaCI but with distinct tempral patterns. The brunt f H fiber activity is immediate and brief, whereas N fiber activity remains high thrughut NaCI stimulatin (22). milride exerted a greater suppressive effect n the respnses f N fibers than n H fibers in bth rats (38) and hamsters (28). dding amilride t NaC1 stimulatin wuld therefre be mre effective by suppressing the tnic respnses f N fibers than amilride pretreatment that fails t suppress the brief respnses f H fibers (28). dditinally, it is als pssible that F344 rats may have a greater prprtin f H fibers r that the threshld fr activating N fibers is higher. milride-sensitive Channels n Taste Cells Nt nly was adding amilride t a stimulating NaCI slutin mre effective in suppressing chrda tympani respnses, it als seemed t be less selective than amilride pretreatment. Studies cnducted mre than 10 years ag in rats fund that amilride pretreatment suppressed the integrated respnses f the whle chrda tympani nerve relatively selectively t NaC1 and LiCI, but nt t KCI, RbCI, and NH4C1 (7,26). Unexpectedly in the present study, amilride additin als suppressed chrda tympani neurn respnses t a stimulating KC1 slutin but t a lesser degree than amilride suppressin f NaCI respnses. milride suppressin f KCI respnses mirrred the same pattern f amilride suppressin f NaC1 respnses. That is, amilride suppressin increased with increasing amilride cncentratin, and decreased with increasing salt cncentratin. The present findings are cnsistent with recent studies shwing: 1) amilride blckade f sdium and ptassium cnductance in islated taste receptr cells frm the frg tngue (1); 2) amilride suppressin f single fiber respnses t KC1 in the rat chrda tympani nerve (38); and 3) amilride suppressin f whle nerve respnses t KCI in the dg chrda tympani nerve (37). Our data indicate that the sdium channel may nt be specific t sdium ins as previusly envisined, r that a cmpnent f the KCI respnse f taste cells may be susceptible t amilride blckade. The latter explanatin seems mre likely insfar as amilride partially suppressed the chrda tympani nerve respnses t KC1, like NaCI. One pssibility is that amilride may blck KCl-stimulated entry f Na + int the receptr cells (41). If this was the case, then the passive sdium current stimulated by KCI may nt be as large as the sdium current generated frm direct NaCI stimulatin. This may explain why amilride suppressin is greater n NaCI than n KC1 respnses. secnd pssibility is that amilride suppresses just the respnses f N

9 MILORIDE INHIBITS TSTE 515 fibers that respnd rbustly t NaCI but may als respnd less t KC1 (38). This may als explain why amilride suppressin is greater n NaC1 than n KC1 respnses. With pretreatment, amilride bathes the receptrs befre salt stimulatin. Fr NaCI t stimulate taste receptrs after amilride pretreatment, sdium ins must displace amilride frm their lcatin in channels n the plasma membrane. The ease with which sdium can displace amilride wuld depend upn hw tightly amiiride binds t the channels. s indicated abve, this may depend als n the duratin f amilride applicatin typically fr lng perids with pretreatment. The respnse envelpe f the chrda tympani nerve t stimulatin depends n hw quickly and efficiently Na + displaced amilride frm the plasma membrane. pparently, the chrda tympani nerve respnse t KC1 may nt require ptassium displacement f amilride, because amilride pretreatment typically fails t suppress KCI respnses. It is mre likely that deplarizatin due t KC1 stimulatin indirectly displaces amilride. When added t the stimulating slutin, taste cells see amilride when they are highly active during stimulatin, nt when the cells are relatively quiescent during baseline. With NaCI stimulatin, amilride and sdium are available t cmpete during sustained tnic activity fr available sdium channel sites n the plasma membrane. When the taste cells are active due t KCI stimulatin, amilride may influence cellular prcesses beynd that invlved in catin detectin and in-amilride cmpetitin, because amilride nt nly blcks sdium channels, but als blcks sdium-hydrgen exchange (4). nd as indicated abve, amilride may als blck passive sdium entry int receptr cells triggered by taste stimuli ther than NaCI (41). CKNOWLEDGEMENTS We thank Beth Basc fr technical supprt. The Natinal Heart, Lung and Bld Institute Grant HL supprted the present research. REFERENCES 1. venet, P. 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