247
Bram Research, 550 (1991) 247-256 © 1991 Elsewer Science Pubhshers B V 0006-8993/91/$03 50 ADONIS 000689939116628L BRES 16628
The effect of amiloride on taste-evoked activity in the nucleus tractus solitarius of the rat Barbara K. Glza and Thomas R. Scott Department of Psychology, Umverstty of Delaware, Newark, DE 19716 (U S A ) (Accepted 18 December 1990)
Key words Amdonde, Nucleus tractus sohtam, Taste, Electrophyslology, Neuron type, Labeled hne
Amdonde is an mhlbttor of passive sodium transport Its application to taste receptors blocks reward sodium current, suppresses sodium-reduced neural actlwty and reduces the percewed intensity of NaC1 We recorded taste-evoked responses of single neurons m the nucleus tractus sohtanus (NTS) of the rat before and after the hngual apphcatlon of amdonde to determine which neurons would be affected, the degree of the effect and the subsequent form of the neural code for sodmm Responses to all 7 st~muh that contamed Na + or Ll+ were suppressed by amdonde Actwlty evoked by the 8 other stimuli was unaltered NTS neurons could be &wded into 4 subsets according to their response profiles Group 1 (salt-sugar), Group 2 (salt), Group 3 (salt-acid) and Group 4 (acid-salt-bitter) The entire effect of amdonde was &scharged on cells m Groups 1 and 2, those m Groups 3 and 4 were unaffected Following amflorlde apphcalaon, the neural code for sodmm and hthmm salts was highly slmdar to those for acids, bitter salts and quinine Thus the actwlty of neurons m Groups 1 and 2 may be responstble for the distinction between 'saltiness' and sour-bitter tastes The results ~mplythat specific receptors are responsible for the recognmon and transduct~on of sodmm salts and that this spec~floty ~s maintained in the peripheral taste nerves to be manifested m the NTS INTRODUCTION
blocks inward sodmm current across frog taste receptor m e m b r a n e s 1 as well as the short circuit Na- or LI-
A m d o n d e (N-amldmo-3,5-dmmlno-6-chloropyrazme carboxamlde) is a potent, reversible inhibitor of passwe
d e p e n d e n t current across the hngual eplthehum of rat 17'18'35, rabbit 34 and dog 26 Accordingly, a m d o n d e
sodium transport whose effectweness spans the animal kingdom A d m i n i s t e r e d orally, a m l l o n d e acts at the distal convoluted tubule and collecting duct of the n e p h r o n to increase s o d m m and bicarbonate excretion but to retain potassmm The resulting dlures~s prowdes the basis for the widespread chmcal use of a m d o n d e m the t r e a t m e n t of hypertension, congestwe heart failure and orrhosls of the hver The apphcatlon of a m d o n d e to the tongue produces results that challenge the classical notion of sodmm transductlon Beldler had proposed that the hngual ep~thehum is ~mpermeable to tastants and that the transduct~on of saltiness results from adsorption of the s o d m m ion to a receptor on the apical m e m b r a n e a However, amdorlde blocks the inward sodmm current, ~mplymg that the m~tlal event revolves passwe transport of Na ÷ across the mucosal surface of the receptor ce1113 14 It ~s now estabhshed that taste cells contain s o d m m transport channels on their apical m e m b r a n e s that are crucial to the ldentlficaUon of sodmm and hthlum ions and to the ultimate perception of saltiness This conclusion derives from the following data (1) a m l l o n d e
causes h y p e r p o l a n z a t l o n and increased resistance across the eplthehum 21'28 (2) Whole nerve 7 12,15,17,2022 and single fiber actwlty 27 to sodium and h t h m m salts m the chorda t y m p a m nerve are suppressed by a m d o n d e (3) H u m a n s whose tongues are treated w~th a m d o n d e experience a reduced perception of saltmess 3° The avallabdlty of a c o m p o u n d that selectively &srupts transductlon of one of the basic taste quahtles prowdes an o p p o r t u m t y to investigate gustatory coding mechanisms We recorded taste-evoked responses of single n e u r o n s in the nucleus tractus sohtarms (NTS) of the rat before and after the hngual apphcatlon of a m d o n d e to determine which n e u r o n s would be affected, the form and extent of the effect, and the resulting neural code for s o d m m - h t h m m salts MATERIALS AND METHODS
Sublects and surgery Subjects were 27 undepnved female Wlstar rats weighing 275-350 g Surgical levels of anesthesia were reduced by intramuscular mjecuon of 100 mg/kg Ketaset followed m 10 mm by 48 mg chloral hydrate, which was subsequently administered as needed
Correspondence B K Glza, Department of Psychology, Umverslty of Delaware, Newark, DE 19716, U S A
248 TABLE I
The sttmulus arrav Stimulus
A bbrevtatton
Human sublecttve taste quahtv 2 ~-25 ~ ~-"
0 01 M 0 03 M 0 10 M 0 30 M 0 10 M 0 50 M 1 00 M 0 03 M 0 01 M 0 01 M 0 01 M 0 10 M 0 10 M 0 10 M 0 20 M
N1 N2 N3 N4 L S G Sa H CI O K Ca M P
weak sweet salty salty salty salty salty sweet sweet salty-sweet sour sour bmer salty-bitter bater meat-hke bland
NaC1 NaCI NaCI NaCI L~CI Sucrose Glucose Sodium saccharin HCI Citric acid Quinine HCI KCI CaCIz MSG Polycose
A t r a c h e o t o m y was performed to permit tracheal clearing and artlficml respiration A n esophageal fistula was implanted to avoid c h e m o s e n s o r y and m e e h a m c a l post-ingestive effects d u n n g gustatory stimulation of the oral cavity, pharynx and e s o p h a g u s T h e rat was m o u n t e d in a non-traumatic head holder and a slender length of perforated vinyl tubing through which a fine stimulus spray could be administered to the entire oral cavity, was placed in its m o u t h and secured The rat's heart rate was monitored and the mterbeat interval maintained at about 180 ms through a d j u s t m e n t s in respiration rate and depth of anesthesm Rectal temperature was maintained between 35 and 37 °C Portions of the occipital and parietal bones were removed and the cerebellum was aspirated to expose the surface of the medulla
Recordmg Activity from single n e u r o n s was isolated by mlcroplpettes (l d = 1 0 Hm, Z = 5 - 1 0 M D ) filled with 1 6 M K citrate Initial coordinates used to locate the NTS were 2 7 m m anterior to the obex, 1 7 m m lateral to the midhne and 1 0 m m ventral to the surface of the medulla The electrode was advanced in 50 ktm steps and neural activity tested until robust gustatory responses were e n c o u n t e r e d T h e preparation was then left undisturbed for approximately 30 mln to permit the tissue to settle around the electrode This increased the probability of achieving the long-term stability necessary to maintain adequate isolation of single neurons over both the pre- and post-amdorlde stimulus series Action potentials were
identified by consistency ot wa'~eform spike a m p h t u d e and the requirement that no two spikes occur within an interval ot less than 1 5 ms Single-unit potentials were amplified filtered and dtsplaw.d on an oscilloscope using conventional recording techniques A direct-coupled 4-channel tape recorder was used to preterit, unit activity, the onset m a r k e r pulse and a voice commentar~ lot later analysis off-hne
Sttmuh and ~ttmulus dehvery A total of 15 stlmuh were employed (see Fable I) The emphasis in th~s array was on Na-Lx salts, but each of the other recognized basic tastes was represented, as were complex combinations of the basic tastes (e g Na saccharin) and stimuli that evoke unique taste perceptions (MSG, polycose) All solutions were prepared ,n dlstdled water except sucrose and glucose to which 5% tap water was added to assure adequate conductivity tot reliable triggering ol a stimulus onset marker Five milliliters of solution was sprayed over the tongue at a rate of 2 ml/s Gustatory-evoked activity was recorded for 5 s following stimulus onset The m o m e n t of stimulus contact with the tongue was marked by a T T L logic device which passed 11-nA current through the rat 9, an a m o u n t two orders of magnitude below the threshold for electric taste 8 2,~ Each tastant was followed by a 20-ml D H z O rinse and by a m i n i m u m rest period of 60 s Stimuli were presented at mtervals of no less than 90 s to prevent possible adaptation effects Additional rinses and rest periods were occasionally required for basehne activity levels to be reestabhshed Stimuli were presented m a quasi-random order with the stipulation that chemicals representing similar taste qualities not be applied consecutively If the neuron remained well isolated at the end of the stimulus series 180 ml of amflonde (0 5 m M ) was sprayed into the m o u t h at a rate of 1 ml/s for 3 m m This was followed b~ a 30-s rest and reapphcatlon ol the stimulus series D u n n g the post-amllonde period all procedures for stimulus application were identical to those used earher except that the amllorlde solution was used for rinses between stlmuh Data analysts D a t a were analyzed off-hne using an I B M - A T c o m p u t e r Spontaneous actwlty was subtracted from the evoked response to yield net spikes/s for the 5-s post-sUmulus period These counts were used to calculate response m a g m t u d e , breadth of t u m n g and average s p o n t a n e o u s rate for all cells prior to a n d following apphcatlon of amllorlde Derived analyses, including calculation of lnterst,mulus and interneuronal correlation matrices, m u l t l d l m e n s m n a l scahng, u m v a n a t e F-tests and post hoe comparisons were all performed with the Systat package s9 Hierarchical cluster analyses, used to Identify groups of cells and stimuli with similar functional properties, were conducted using the clustan routine on an I B M 3090 computer
Total
u-q Pre t~R Post
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Fig 2 Mean response profiles across all n e u r o n s to the stimulus array prior to (dashed hne) and following (solid hne) lingual application of amflorlde Spontaneous activity h a s been subtracted from the total response to each stimulus Abbreviations are from Table I X mean response across all stimuli, * P ~ 0 05
249 Summary staustlcs mcludmg calculation of mean spontaneous rate, breadth of tuning and evoked rates were obtamed for each neuronal cluster as they had been for the entire population of cells Finally net spikes for each of 50 consecutwe 100-ms bins were averaged across umts to gtve post-sttmulus t~me Mstograms (PSTHs) for all sttmuh pre- and postamdonde From these, temporally based correlation matnces were calculated and mult~dtmens~onal spaces were generated based on the relative s~mtlartty of responses t~me courses
0 328 0 392 0 456 0 519
0 583 0 647 0711 0 775
RESULTS
0 838 0 902
General fmdmgs A c r o s s all cells r e c o r d e d in the NTS, stgnlficantly less acttvity was e v o k e d by sodium and lithium salts, M S G and sodium s a c c h a n n following amllorlde The magnitude of the decrease - - 47 1% to 0 1 M NaCI - - is consistent with that o b t a m e d from whole nerve chorda t y m p a n l responses in several species 7'i2J5'17-2°22 mmiloride did not affect the activity e v o k e d by sugars, acids or bitter stimuli M o r e revealing, however, was the effect of a m t l o n d e on subtypes of NTS taste cells Neurons could be categorized into 4 groups b a s e d on their response profiles to the entire stimulus array before the apphcation of amtlorlde (see division into neuron types below) G r o u p 1 (salt-sugar) showed g o o d sensitiwty to Na-Lt salts plus a side b a n d of responsiveness to the sugars G r o u p 2 (salt) neurons gave the same strong response to Na-Li salts, but were not effectively driven by any other stimulus G r o u p 3 (salt-acid) cells were equally sensitive to NaCI and LiCI but also r e s p o n d e d well to HCI and citric acids and to CaCI 2 G r o u p 4 (acid-salt-bitter) also r e s p o n d e d well to NaCI and LICI, but showed exquisite sensitivity to the two acids and a side b a n d of responstveness to the 3 bitter stimuli Thus, whtle the 4 groups showed roughly equal sensitivity to Na-LI salts, their full profiles p e r m i t t e d statistically reliable discrimination a m o n g t h e m T h e effect of amlloride was to suppress salt responses in G r o u p s 1 and 2 profoundly while leaving those in G r o u p s 3 and 4 unaffected
Spontaneous acttvtty and response cnterton The criterion for an e v o k e d response was a change in neural actlvlty of 1 65 S D ( P < 0 05, one-tailed) from m e a n s p o n t a n e o u s rate for each cell, sustained for 5 s M e a n s p o n t a n e o u s rate before the application of amiloride was 11 7 + 11 0 spikes/s, a value that did not differ significantly among the 4 subgroups (F3 32 = 0 429, non-significant) A m i l o n d e r e d u c e d the overall spontaneous activity by only a non-significant 1 3 splkes/s ( F 3 32
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PRE AIdlLORIDE uNrr5 1--37 (NET 5 SEC) Fig 3 Results of a d u s t e r analysts deptcted as a d e n d r o g r a m m whtch n e u r o n s are related to one a n o t h e r according to the s~mllanty of thetr response profiles prior to a m d o n d e apphcatton
= 0 837), no neuronal subgroup was slgntficantly suppressed, though G r o u p 2 (salt) n e u r o n s e x p e n e n c e d a reduction that a p p r o a c h e d significance (see Fig 1)
Evoked acttvlty Breadth of responstveness NTS cells showed typically b r o a d responsiveness across the sttmulus array p n o r to amiloride a p p h c a t i o n O f 555 total stimulus apphcatlons (37 neurons × 15 sttmuh) 78 9 % fulfilled the c n t e n o n for excitation, 0 4% for inh]bmon, and 20 7 % e v o k e d no response Following a m l l o n d e , t h e r e was a non-significant decline to 69 9% excitation, whtle 1 1% of the stimulus apphcations caused inhibition and 29 0 % no response Across all cells, the adminlstratton of amlloride did not result in sigmficant changes in b r e a d t h of responsiveness according to 3 s t a n d a r d measures the p r o p o r t i o n of stlmuh to which each n e u r o n r e s p o n d e d , the p r o p o r t i o n of neurons r e s p o n d i n g to each stimulus, or the breadth-of-tuning ( B O T ) metric *'36 H o w e v e r the n u m b e r of cells that r e s p o n d e d to Na-Li salts declined by 23 0% following amiloride The B O T metric p r o v e d m o r e r e v e a h n g when applied to the responses of neuronal subgroups T h e m e a n B O T was 0 79 pre- and 0 83 p o s t a m i l o r t d e (F] 32 = 3 796, n s ) But while most cells in G r o u p s 1, 3 and 4 showed marginal decreases in b r e a d t h due to the reduction of the sodium response c o m p o n e n t , G r o u p 2 (salt) neurons experienced a m a r k e d increase from 0 68 to 0 83 ( F 1,32 = 17 242, P < 0 001), a result that reflects their loss of responsiveness to the stimulus that c o n f e r r e d specificity
* The breadth-of-tuning metric prov2des an index of the dtstrlbutton of a cell's response across the 4 prototyplcal stlmuh It is calculated according to the formula H = -k~; p,logp, where H = breadth-of-tuning coefftcmnt, k = a scahng constant, whmh is 1 661 for 4 sttmuh, p, = the proportion of the neuron's response devoted to any one stimulus The value of H ranges from 0 00 for a neuron that responds to only one of the prototyplcal stlmuh, to 1 00 when the response ~s equal to all 4
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Fig 4 Mean response profiles of each subgroup of neurons prior to (dashed hne) and following (solid hne) amdonde apphcaUon Abbrevlauons are from Table I X, mean response to all stimuh, *, P < 0 05 (reprinted from Scott and Glza, Science, 249 (1990) 1585-1587 with permission Copyright 1990 by the AAAS)
General effect o f amdortde on response rate Activity e v o k e d by the 7 c o m p o u n d s containing Na ÷ or L1÷ d e c h n e d by 33 to 51% ( P < 0 003 in all cases) following the a p p h c a t l o n of a m t l o n d e , while responses to all o t h e r sttmuh were unaffected (Fig 2) W e a k e r NaCI solutions were inhibited p r o p o r t i o n a t e l y m o r e than those that were c o n c e n t r a t e d , and responses to the halide salts were s u p p r e s s e d m o r e than those of M S G and Na saccharin These effects are m o r e strIkmg when neural groups are defined and considered m d e p e n d e n t l y , as shown below Division mto neuron types T h e r e are many m o r p h o logical and functional criteria by which neurons may be c a t e g o n z e d 6 1 1 , 1 6 , 3 7 38 O u r interest m thts study was the influence of a m t l o n d e on taste-evoked activity Thus an a p p r o p r i a t e index for neuronal c a t e g o n z a t t o n is the response profile of a cell, both before amlloride apphcation to help organize the system in anticipation of the amtlortde, and afterward to d e t e r m i n e how taste cells are r e a h g n e d by its effects The net (total mmus spontaneous) discharges e v o k e d by each stimulus in 5 s constitutes the 15-point response profile for each of the 37 neurons before a m f l o n d e is a d m i n i s t e r e d Pearson p r o d u c t - m o m e n t c o r r e l a t m n coefflctents are then calculated between all pairs of profiles (n = 37 x 36 = 666 coefficients), providing a matrLx of relative functional slmIlanty among all cells This matrix may be subjected to a cluster analysis 4° the result of which takes the form of a d e n d r o g r a m (Fig 3) Neurons are n u m b e r e d in the o r d e r in which they were isolated Pairs of cells are Interconnected at the level of correlation between their response
profiles, groups are fused at the m e a n level of correlation between their constituent m e m b e r s in an l t e r a t w e process until all neurons in the sample are connected The suggestion from the p r e - a m t l o r l d e d e n d r o g r a m shown in Fig 3 is that our neural sample m a y be classified into 4 statistically dtstmct clusters that m a y be t r e a t e d independently m further analyses** B e n e a t h each cell n u m b e r in Fig 3 ts labeled the stimulus that e v o k e d the greatest response, followed by any o t h e r stimulus that elicited at least 80% of that m a x i m u m The left-hand cluster ( G r o u p 1) includes cells 1-37 (n = 9), whose p n m a r y sensitivity was to NaC1 (N)" but which also r e s p o n d e d relatively well to sugars and s o d i u m sacchann (S) This group includes response profiles that are fully mtercorrelated at a level of + 0 73 Next to it ts G r o u p 2 c o m p o s e d of cells 10-33 (n = 10) which r e s p o n d e d almost exclusively to NaCl and whose profiles are c o r r e l a t e d at a level of + 0 69 or a b o v e G r o u p 3 encompasses cells 4 - 2 6 (n = 8), all of which are also connected at a level of + 0 69 or a b o v e , this group ts characterized by nearly equal responses to NaCl and the acids G r o u p 4 includes cells 6 - 2 8 (n = 9), all lntercorrelated at + 0 71, and showmg p r e d o m i n a n t responsweness to the acids with strong sldebands to NaCl and to bitter substances In all 4 groups, the m e a n response to 0 1 M NaCl and LtCl was roughly equivalent at nearly 40 splkes/s Cell 17 had a profile that c o r r e l a t e d p o o r l y with all others, and so was excluded from the following analyses Effect o f armlonde by subgroup The application of
** We do not maintain that we have found exactly 4 gustatory neuron types The cluster analys~s also permits a stausncal d~wsion into just two groups (cells 1-17 and cells 4-28) or, conversely, into a large number of more exclusive clusters We take 4 to be a convenient and manageable number of rather clearly defined groups
251 amllorlde to the tongue had sharply contrasting effects on the responsiveness of neurons of different subgroups to Na-L1 compounds (Fig 4) The sensitivity of cells in Group 1 (salt-sugar) declined to the 4 concentrations of NaCl by a mean of 79%, to 0 1 M LIC1 by 74% and to 0 03 M Na saccharin by 30% (the response here was presumably sustained in part by the sweetness of saccharin in these sugar-sensitive cells) Their responses to all other stimuli were unaffected Group 2 (salt) cells showed a mean decline of 68% to the 4 concentrations of NaCl, 75% to 0 1 M LiCl, 58% to 0 1 M MSG and 52% to 0 03 M Na saccharin In contrast, the salt sensitivity of cells in Groups 3 and 4 was unaffected by amIloride Group 3 (salt-acid) neurons showed only an increase In response to citric acid, while Group 4 (acid-salt-bitter) cells experienced small but significant reductions to MSG and sucrose When the postamllonde response of each neuron to 0 1 M NaCl was expressed as a percentage of the pre-amIloride response, the difference among neuron groups was obvious (Fig 5) There was almost no overlap between the effect of amdoride on the first two versus the latter two groups, despite the fact that the mean pre-amdoride response to Na-LI salts was similar In all groups Th~s point Is dlustrated in Fig 6 which contains raw records of the responses of a representative cell from each group to 0 1 M NaCl before and after amilonde apphcat~on Thus, the broad response profile, rather than the response to Na-LI salts alone, provides nearly perfect predlctabdlty of which taste neurons in the central nervous system will be affected by interfenng with a transductlon mechamsm for Na and L1 ions Taste quahty Just as a 15-point profile across stimuli could be generated to represent the response pattern of each of the 37 neurons, so a 37-point profile across neurons may represent the response pattern evoked by each stimulus The correlation coeffioent between any pair of profiles is taken as an index of the relative similarity of the two taste qualmes they represent Alterations m the taste quality of a stimulus, occasioned here by the apphcatlon of amiloride, would appear as changes m the correlation between its profile and those of all other stimuh In Fig 7 we have plotted the correlaUons between the profile generated by 0 1 M NaC1 and those of the 14 other stimuli, both before (left column) and after amiloride Three distinct results emerge from this analysis First, 0 1 M NaCl largely maintained its close relationship with 0 1 M LICI, and the 3 remaining concentrations of NaC1, the mean of these 4 correlations being +0 92 pre- and +0 86 postamilorlde This minor reduction stems from the response suppression that typically yields lower coeffloents It is expected that these coeffioents should remain rather constant, for
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Fig 5 Response to 0 1 M NaCI following amflonde apphcatlon expressed as a percentage of each neuron's response before amdonde Valuesbelow zero indicate mhlbltory responses following amdonde Arrows mark the neurons whose mdwtdual responses are depicted m Fig 6 (GI, cell 21, G2, cell 11, G3, cell 9, G4, cell 21)
any influence of amllonde should be exerted across all 5 of the Na-LI salts we used Secondly, there was an increase In stmdanty between the profile of 0 1 M NaCl and those of the 5 sumuh that humans characterize as bitter-sour KCI, CaC12, quinine HC1, HCI and citric acid The mean of these correlations rose with amllonde application from +0 69 to +0 86, the same mean coefficient that 0 1 M NaCl had with other Na-Ll salts This result derives from the selective loss of responsiveness to Na-LI salts in neuronal Groups 1 and 2 The remaining activity evoked by NaCl and LICI is m Group 3 and 4 cells where sensitivity to bitter and sour stimuli is also high Thus there is increased covanance between the neurons that respond to Na-Ll salts and the bitter chemicals and acids, and so higher correlations between their profiles Finally the coefficient between 0 1 M NaCl and the unique tastes of MSG (meat-like) and polycose (starchy) was reduced moderately while its correlation with stimuli that humans describe as sweet - - sucrose, glucose, Na sacchann - - plunged from a mean of +0 68 to +0 26 The latter occurred because Group 1 neurons that shared a sensitivity to Na-LI salts and sugars, lost that common bond to the effects of amllorlde, and with it the covarlance that permitted at least moderate correlations Taken together, these results imply that the tastes of NaCl and LICI became predominantly sour-bitter followmg amiloride The matrix of correlations between all pairs of stimulus profiles (n = 15 x 1 4 - 2 = 105) may be used to create a mulUdimensional space In which the relative similarity of stimuli is proportional to their proximity Such a space,
252
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Fig 6 Responses of a representative neuron from each group to 0 I M NaCI prior to (left) and following (right) hngual apphcation of amdonde The evoked (total minus spontaneous) response declined by 92% both in cell 21 (GI) and m cell 11 (G2) while tt rose 1% m cell 9 (G3) and decreased by 6% m cell 13 (G4)
derived from neural responses before a m d o n d e apphcatlon, ~s shown m Fig 8 A In It may be seen a typical distribution of stimuli, with sugars and bitter-sour st~muh located at opposite extremes, Na-L1 salts and M S G form a tight central group, while polycose is d,stmct from all o t h e r sUmuh The space m Fig 8B is g e n e r a t e d from responses r e c o r d e d after a m d o n d e t r e a t m e n t As exp e c t e d from the preceding analysis, the 5 Na-L] salts have m o v e d to the sour-b,tter extreme, leaving only M S G m
the p o s m o n they formerly occupied It seems a reasonable mference that if the Na-LI salts r e p r e s e n t the perception of 'saltiness' m the n o r m a l l y funcUonmg taste system (Fig 8A) then this quahty has been lost with a m d o n d e (Fig 8B) T h e r e f o r e , with the Na-Ll response suppressed m G r o u p 1 and 2 neurons, 'saltiness' a p p e a r s to be e h m m a t e d although a p p r o x i m a t e l y 50% o f the total activity e v o k e d by these salts remains 'Saltiness' may be coded by neurons whose profiles emphasize Na-L! re-
253
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Fig 7 Correlations between the profile evoked by 0 1 M NaCI and that of each remaining stimulus before (pre) and following (post) amllonde application
sponsweness and exclude sensmvaty to other non-sweet stamuh These cells may be served by receptors that use
a passive transport channel (susceptable to amflonde blockade) as a transductaon mechamsm, as opposed to receptors lnnervatmg those neurons that respond broadly to non-sweet chemicals, including NaCI and LICI These latter neurons may be responding to the halogen anaon of the NaCI molecule 15 Temporal analysts The correlataons among stamulus profiles above are based on the total response of each cell to every sUmulus, summed over the 5-s post-stamulus recordmg period Thus the tame course over whach the response develops is not considered To analyze temporal propertaes, each response was dwaded into fifty 100-ms bans and summed across neurons for a gwen stamulus Thas represents the temporal response profile for that chemacal Correlataons among all pmrs of profiles (n = 15 x 1 4 - 2 = 105) could be calculated and multadlmensaonal spaces - - now based on temporal propertaes - - generated as before Spaces derwed from responses before (Fig 9A) and after (Fag 9B) amdonde apphcatlon are remarkably samdar, and mdacate that the severe suppressaon of Na-La responses occurs m a temporally uniform manner In both spaces, stamuh that are characterized by hagh phasac and relatwely low tomc actwaty are posatloned toward the left, while those with low phasic-
A I
2
P N
HN1C~
2
~
H N
3
G
s H ~'I Sa
Fig 8 Three-dimensional space representing the relative s,mdarmes of response profiles evoked by each stimulus before (A) and after (B) amdorlde Abbreviations are from Table I The axes are not defined (reprinted from Scott and Giza, Scwnce, 249 (1990) 1585-1587, with permtsseon Copyright 1990 by the AAAS)
B
I
F~g 9 Three-dimensional spaces representing relative similarities among the time courses of evoked responses before (A) and following (B) amllonde application Abbreviations are from Table I The axes are not defined
254 to-tomc ratios are s~tuated at the right DISCUSSION
Imphcattons for gustatory codmg The most striking findings of this study of NTS taste cells are (1) the effects of a m d o r l d e on responsweness to NaCI and LIC1 are either profound or minimal, d e p e n d mg on the cell, and (2) the response profile of any neuron permits a confident predtct~on of which result wdl be o b t a i n e d for that cell In G r o u p s 1 and 2, 15 of 19 cells had their Na-L1 responsweness suppressed by m o r e than 7 5 % , m G r o u p s 3 and 4, 15 of 17 were suppressed by less than 25% This provides clear evidence that r e c e p t o r cells using a passwe s o d m m transduction mechanism relay through p e r i p h e r a l nerves to ~dentffmble subgroups of CNS taste cells M o r e o v e r , when this mechamsm is b l o c k e d by a m d o n d e , the r e m a m m g actw~ty e v o k e d by Na-L1 salts is highly slmdar to that e l i o t e d by a o d s and b~tter c o m p o u n d s This ~mphes that the actw~ty of G r o u p 1 and 2 neurons ~s responsible for the p e r c e w e d distraction between 'saltiness' and sour-bitter taste and so argues for the notion that certain taste cells perform specific coding functions A m l l o n d e has been r e p o r t e d to cause an increase m m e m b r a n e resistance and a hyperpolanzat~on of 20 mV m a subset of rat taste r e c e p t o r cells 2 3 These receptors, which constitute about 25% of the population, should subsequently be responsive only to st~muh that cause s u f f t o e n t d e p o l a r i z a t i o n to o v e r c o m e this suppresswe influence The i n t e n s i t y - r e s p o n s e functions we r e c o r d e d from G r o u p 1 and 2 cells m the NTS across 4 concentrations of NaC1 fulfill thts expectation p o s t a m d o n d e response functions rise monotonically with stimulus intensity, though actwlty levels are suppressed some 75% from those r e c o r d e d m the u n a d u l t e r a t e d system*** F u r t h e r m o r e , ff a r e c e p t o r cell is h y p e r p o l a n z e d by a m d o r l d e its responsiveness to all stlmuh should be suppressed, regardless of the mechanism by which it is n o r m a l l y d e p o l a r i z e d Thus the actwlty aroused m that cell by sugars, a o d s , bitter salts and alkaloids should be r e d u c e d as much as that to NaCI and LICI The fact that responses m the NTS to all stimuli that did not contain Na or Ll ions were unaffected by a m d o n d e apphcatlon ~mphes that the transductlon of these sUmuh ~s accomphshed entirely by r e c e p t o r cells that are not a m d o n d e sensitwe Thus there ~s a sharp distmct~on, estabhshed at the r e c e p t o r level and m a i n t a i n e d in peripheral nerves to be manifested in the m e d u l l a amdorlde-sensltwe receptors recognize the presence of Na + and L~+ but do not
part~opate m the transduct~on of any o t h e r class ot tast~ stimulus These receptors project p r e d o m i n a n t l y to neurons we have included m G r o u p s t and 2 which are thus r e n d e r e d h y p o r e s p o n s w e by a m l l o n d e G r o u p 1 neurons presumably receive input as well from a separate subset of receptors that transduce sugars, and these responses remain following a m d o n d e t r e a t m e n t G r o u p 2 cells are largely deprived of their only m a j o r source of lnnervatlon and may r e s p o n d just to residual activity from sodmm receptors and perhaps to m i n o r mputs trom others Thus we are led to conclude that coding channels exist within the taste system, a p o s m o n that is d e v e l o p e d more tully elsewhere ~
The taste quahty of NaCl Despite the increase m simllartty b e t w e e n Na-Ll salts and stlmuh that humans label sour or bitter, fireddeprived rats are r e p o r t e d to accept NaCI m o r e wdhngly after their tongues are treated with a m d o n d e 5 lo This would imply an i m p r o v e m e n t m h e d o m c quahty, m contrast to the tmpllcatlons of o u r electrophystologlcal results H o w e v e r , this behavtor ts only seen when NaCI ~s p r e s e n t e d at concentrations of 0 3 and 0 5 M The aversweness of these stimuli would be a t t e n u a t e d by amllorlde-lnduced suppresston, such that the net result of changes m p e r c e w e d intensity (toward m o r e acceptable levels) and quahty ( p r e s u m a b l y t o w a r d those that are less acceptable) are unpredtctable I n c r e a s e d acceptance does not result from a m l l o n d e t r e a t m e n t when the stimulus is a m o r e palatable 0 1 M NaCI Thus, there is r o o m within the bounds of our data to a c c o m m o d a t e the behavioral findings In support of the notion of a shift in quahty, Bernstem reports that s o d m m - d e p r i v e d rats, whose physiological need renders 0 5 M s o d m m p a l a t a b l e , are less wdhng to consume th~s solution when they are p r e t r e a t e d w~th a m d o r t d e T h e r e f o r e , when intensity ~s not a deterrant, the p r e s u m e d shift of the quahty ehc~ted by NaCI t o w a r d m o r e hedomcally n e g a t w e tastes is behaviorally manifest
The tastes of MSG and polycose A m l l o r t d e suppressed responses to M S G by httle more than half as much as those to NaCI and L1CI H o w e v e r , the taste quahty of M S G r e m a i n e d firm, even as those of NaCI and L~C1 m o v e d t o w a r d bitter-sour c o m p o u n d s (Fig 8) This is quite slmtlar to the effect on Na saccharin, whose responses were suppressed to the same degree as those of M S G and which also m a i n t a i n e d its p o s m o n m the stimulus space after a m d o r i d e Just as Na saccharin has a d o m i n a n t quahty (sweet) to sustain Its
*** It is puzzhng, however, that spontaneous actlvuy in these same cells, which might be expected to be most suppressed by a severe hyperpolanzatlon of the receptors that serve them, was reduced by only 2% (Group l) and 35% (Group 2)
255 l d e n t a y when saltmess has been blocked, so presumably
taste ~s w h e t h e r the afferent signal ~s read across the
does M S G ( U m a m 0 41 Thus these data provide evidence
entire population of n eu r o n s or ~s restricted to a channel
for a taste quahty for M S G that transcends saltiness, and
of activity most sahent to the transmission of a particular
which ~s not transduced p n m a r d y through passive s o dm m
quahty
channels
nism through a m d o n d e apphcat~on has r e v e a l e d deficits
Polycose has also b e e n touted as the p r o to ty p e for the i n d e p e n d e n t taste quahty 'starch '32 In support of th~s,
N TS that p r o m o t e the latter v~ew
T h e d~sabhng of a s p e o f i c transduct~on mecha-
m the responses of ~denUfiable neural subgroups m the
polycose ehc~ted an actlwty profile that was poorly correlated w~th those of all accepted prototypes and which was unaffected by amllortde
Conclusion A m a j o r ~ssue m d~scuss~ons of the neural code for
REFERENCES 1 Avenet, P and Lmdemann, B , Amflonde-biockable sodmm currents m isolated taste receptor cells, J Membr Blol, 105 (1988) 245-255 2 Behe, P, DeSlmone, J A , Avenet, P and Llndemann, B , Patch-clamp recording from taste buds of mamtamed eplthehal polanty a novel approach, ISOT X, 72 (1989) 3 Behe, P, DeSlmone, J A , Avenet, P and Lmdemann, B , Patch-clamp recordings from ~solated taste buds responses to sacchann and amdonde, ISOT X, 73 (1989) 4 Beldler, L M , A theory of taste sumulatlon, J Gen Phystol, 38 (1954) 133-139 5 Bernstem, I L and Hennessy, C J , Amdorlde-sensltwe sodium channels and expression of sodium appetite m rats, Am J Phystol, 253 (1987) R371-R374 6 Boudreau, J C and Alev, N , Classification of chemoreceptwe tongue umts of the cat genlculate ganghon, Brain Research, 54 (1973) 157-175 7 Brand, J G , Teeter, J H and Sliver, W L , Inhlbmon by amdonde of chorda tympam responses evoked by monovalent salts, Brain Research, 334 (1985) 207-214 8 Bujas, Z , Electric taste In L M Beldler (Ed), Handbook of Sensory Physiology, Vol IV, Spnnger, Berhn, 1971, pp 180199 9 Chang, F-C T and Scott, T R , Gustatory stimulus dehvery m the rodent, Chem Senses, 9 (1984) 91-96 10 Contreras, R J , Farnum, E K and Bird, E , Amflonde alters behaworal and neural gustatory responses to salt solutions, Chem Senses, 10 (1985) 430 11 Davis, B J and Jang, T , A golgl analysis of the gustatory zone of the sohtary tract m the adult hamster, J Comp Neurol, 278 (1988) 383-396 12 DeSlmone, J A and Ferrell, F, Analysts of amdonde mhlbmon of chorda tympam taste response of rat to NaCI, Am J Physlol, 249 (1985) R52-R61 13 DeS~mone, J A , Heck, G L and DeSimone, S K , Actwe ion transport m dog tongue a possible role in taste, Sctence, 214 (1981) 1039-1041 14 DeSlmone, J A , Heck, G L , Mlerson, S and DeSlmone, S K , The actwe ion transport properties of canine hngual epttheha in v~tro Imphcat~ons for gustatory transduct~on, J Gen Physlol, 83 (1984) 633-656 15 Formaker, B K and Hdl, D L , An analysis of residual NaCI taste response after amdonde, Am J Physiol, 255 (1988) R1002-R1007 16 Frank, M E , Contreras, R J and Hettmger, T P, Nerve fibers sens~twe to ~omc taste sUmuh m chorda tympam of the rat, J Neurophysiol, 50 (1983) 941-960 17 Heck, G L , Mlerson, S and DeSlmone, J A , Salt taste transductlon occurs through an amllonde-sensit~ve sodium trans-
Acknowledgements We thank Robert F Antonucci and Katherine T Spence for assistance m data collection and Judith A Fmgerle and Barbara Parker for preparation of the manuscript This research was supported by NIH Grant DK30964 and by the Campbell Institute
port pathway, Science, 223 (1984) 403-405 18 Heck, G L , Welter, M E and DeSlmone, J A , Simultaneous recordings of the transeplthehal hngual potential and integrated neural response of the rat, Chem Senses, 10 (1985) 427-428 19 Hellekant, G , DuBolS, G E , Roberts, T W and Van der Wel, H , On the gustatory effect of amllonde m the monkey (Macaca mulatta), Chem Senses, 13 (1988) 89-93 20 Herness, M S , Effect of amdonde on bulk flow and lontophoretlc taste stlmuh m the hamster, J Comp Physlol, A160 (1987) 281-288 21 Herness, M S , Effect of amdonde on iontophoretlc and chemical stimulation in hamster and frog, IX Int Symp Olf Taste, 541 (1986) 22 Hill, D L and Bour, T C , Addmon of functional amdondesens~tive components to the receptor membrane a possible mechamsm for altered taste responses during development, Develop Brain Res , 20 (1985) 310-313 23 Jakmovlch, W, J r , Sugar taste reception in the gerbd In D W Pfaff (Ed), Taste, OIfacUon and the Central Nervous System, Rockefeller Unlv, New York, 1984, pp 65-91 24 Lee, C K , Carbohydrate sweeteners stuctural requirements for taste, World Rev Nutr Diet, 33 (1979) 142-197 25 McBurney, D H , Smith, D V and Shlck, T R , Gustatory cross adaptlon sourness and bitterness, Percep Psychophys, 11 (1972) 228-232 26 Mlerson, S , Heck, G L , DeSlmone, S K , Blber, T U L and DeSimone, J A , The ~dentity of the current earners m camne lingual eplthehum in vitro, Blochlm Blophys Acta, 816 (1985) 283-293 27 Nmomlya, Y and Funakoshl, M , Amflonde inhibition of responses of rat single chorda tympam fibers to chemical and electrical tongue stlmulauons, Brain Research, 451 (1988) 319-325 28 Okada, Y, Mlyamoto, T and Sato, T , Contribution of the receptor and basolateral membranes to the restmg potential of a frog taste cell, Jap J Physlol, 36 (1986) 139-150 29 Pfaffmann, C and Pntchard, T C , Ion specificity of electric taste In H van der Starre (Ed), Olfacuon and Taste, Vol VII, IRL, London, 1980, pp 175-178 30 Schiffman, S S , Lockhead, E and Maes, F W , Amdonde reduces the taste intensity of Na and L~ salts and sweeteners, Proc Natl Acad Sct U S A , 80 (1983)6136-6140 31 Schiffman, S S , McElroy, A E and Enckson, R P, The range of taste quahty of sodmm salts, Phystol Behav, 24 (1980) 217-224 32 Sclafanl, A , Carbohydrate taste, appetite and obesity an overview, Neuroscl Biobehav Rev, 11 (1987) 131-153 33 Scott, T R and Giza, B K , Coding channels in the taste system of the rat, Sctence, 249 (1990) 1585-1587 34 Simon, S A , Robb, R and Garvln, J L , Eplthehal responses of rabbit tongues and their involvement m taste transduct~on, Am
256
J Phystol, 251 (1986) R 5 9 8 - R 6 0 8 35 Simon S A , Robb, R and Schlffman, S S , Transport pathways m rat hngual e p l t h e h u m Pharmacol Btochem Beh , 29 (1988) 257-267 36 Smith, D V and Travers J B , A metric for the breadth ot tuning of gustatory neurons, Chem Senses, 4 (1979) 215-229 37 Travers, J B , Efferent proJections from the anterior nucleus of the sohtary tract of the hamster Bram Research 457 (1988) 1-11 38 Whztehead, M (~ , A n a t o m y of the gustatory system m the
h a m s t e r svnaptology of laclal afferent terminals in the sohtar~, nucleus, J Comp Neurol 244 (1986) 72-85 39 Wilkinson L SYSTAT The 7vstemforStatlstt~s S Y S T A T I n t Evanston, IL 1988 40 Wlshart D , Clustan User Manual, C o m p u t i n g Laboratory Umvers,ty of St A n d r e w s St A n d r e w s 1987 41 Yamaguchl, S , F u n d a m e n t a l properties ol U m a m l m h u m a n taste sensation In Y K a w a m u r a and M R Kare ( E d s ) Umarm A Basic Taste, D e k k e r New York 1987 pp 41-73
Bram Research, 550 (1991) 247-256 © 1991 Elsewer Science Pubhshers B V 0006-8993/91/$03 50 ADONIS 000689939116628L BRES 16628
The effect of amiloride on taste-evoked activity in the nucleus tractus solitarius of the rat Barbara K. Glza and Thomas R. Scott Department of Psychology, Umverstty of Delaware, Newark, DE 19716 (U S A ) (Accepted 18 December 1990)
Key words Amdonde, Nucleus tractus sohtam, Taste, Electrophyslology, Neuron type, Labeled hne
Amdonde is an mhlbttor of passive sodium transport Its application to taste receptors blocks reward sodium current, suppresses sodium-reduced neural actlwty and reduces the percewed intensity of NaC1 We recorded taste-evoked responses of single neurons m the nucleus tractus sohtanus (NTS) of the rat before and after the hngual apphcatlon of amdonde to determine which neurons would be affected, the degree of the effect and the subsequent form of the neural code for sodmm Responses to all 7 st~muh that contamed Na + or Ll+ were suppressed by amdonde Actwlty evoked by the 8 other stimuli was unaltered NTS neurons could be &wded into 4 subsets according to their response profiles Group 1 (salt-sugar), Group 2 (salt), Group 3 (salt-acid) and Group 4 (acid-salt-bitter) The entire effect of amdonde was &scharged on cells m Groups 1 and 2, those m Groups 3 and 4 were unaffected Following amflorlde apphcalaon, the neural code for sodmm and hthmm salts was highly slmdar to those for acids, bitter salts and quinine Thus the actwlty of neurons m Groups 1 and 2 may be responstble for the distinction between 'saltiness' and sour-bitter tastes The results ~mplythat specific receptors are responsible for the recognmon and transduct~on of sodmm salts and that this spec~floty ~s maintained in the peripheral taste nerves to be manifested m the NTS INTRODUCTION
blocks inward sodmm current across frog taste receptor m e m b r a n e s 1 as well as the short circuit Na- or LI-
A m d o n d e (N-amldmo-3,5-dmmlno-6-chloropyrazme carboxamlde) is a potent, reversible inhibitor of passwe
d e p e n d e n t current across the hngual eplthehum of rat 17'18'35, rabbit 34 and dog 26 Accordingly, a m d o n d e
sodium transport whose effectweness spans the animal kingdom A d m i n i s t e r e d orally, a m l l o n d e acts at the distal convoluted tubule and collecting duct of the n e p h r o n to increase s o d m m and bicarbonate excretion but to retain potassmm The resulting dlures~s prowdes the basis for the widespread chmcal use of a m d o n d e m the t r e a t m e n t of hypertension, congestwe heart failure and orrhosls of the hver The apphcatlon of a m d o n d e to the tongue produces results that challenge the classical notion of sodmm transductlon Beldler had proposed that the hngual ep~thehum is ~mpermeable to tastants and that the transduct~on of saltiness results from adsorption of the s o d m m ion to a receptor on the apical m e m b r a n e a However, amdorlde blocks the inward sodmm current, ~mplymg that the m~tlal event revolves passwe transport of Na ÷ across the mucosal surface of the receptor ce1113 14 It ~s now estabhshed that taste cells contain s o d m m transport channels on their apical m e m b r a n e s that are crucial to the ldentlficaUon of sodmm and hthlum ions and to the ultimate perception of saltiness This conclusion derives from the following data (1) a m l l o n d e
causes h y p e r p o l a n z a t l o n and increased resistance across the eplthehum 21'28 (2) Whole nerve 7 12,15,17,2022 and single fiber actwlty 27 to sodium and h t h m m salts m the chorda t y m p a m nerve are suppressed by a m d o n d e (3) H u m a n s whose tongues are treated w~th a m d o n d e experience a reduced perception of saltmess 3° The avallabdlty of a c o m p o u n d that selectively &srupts transductlon of one of the basic taste quahtles prowdes an o p p o r t u m t y to investigate gustatory coding mechanisms We recorded taste-evoked responses of single n e u r o n s in the nucleus tractus sohtarms (NTS) of the rat before and after the hngual apphcatlon of a m d o n d e to determine which n e u r o n s would be affected, the form and extent of the effect, and the resulting neural code for s o d m m - h t h m m salts MATERIALS AND METHODS
Sublects and surgery Subjects were 27 undepnved female Wlstar rats weighing 275-350 g Surgical levels of anesthesia were reduced by intramuscular mjecuon of 100 mg/kg Ketaset followed m 10 mm by 48 mg chloral hydrate, which was subsequently administered as needed
Correspondence B K Glza, Department of Psychology, Umverslty of Delaware, Newark, DE 19716, U S A
248 TABLE I
The sttmulus arrav Stimulus
A bbrevtatton
Human sublecttve taste quahtv 2 ~-25 ~ ~-"
0 01 M 0 03 M 0 10 M 0 30 M 0 10 M 0 50 M 1 00 M 0 03 M 0 01 M 0 01 M 0 01 M 0 10 M 0 10 M 0 10 M 0 20 M
N1 N2 N3 N4 L S G Sa H CI O K Ca M P
weak sweet salty salty salty salty salty sweet sweet salty-sweet sour sour bmer salty-bitter bater meat-hke bland
NaC1 NaCI NaCI NaCI L~CI Sucrose Glucose Sodium saccharin HCI Citric acid Quinine HCI KCI CaCIz MSG Polycose
A t r a c h e o t o m y was performed to permit tracheal clearing and artlficml respiration A n esophageal fistula was implanted to avoid c h e m o s e n s o r y and m e e h a m c a l post-ingestive effects d u n n g gustatory stimulation of the oral cavity, pharynx and e s o p h a g u s T h e rat was m o u n t e d in a non-traumatic head holder and a slender length of perforated vinyl tubing through which a fine stimulus spray could be administered to the entire oral cavity, was placed in its m o u t h and secured The rat's heart rate was monitored and the mterbeat interval maintained at about 180 ms through a d j u s t m e n t s in respiration rate and depth of anesthesm Rectal temperature was maintained between 35 and 37 °C Portions of the occipital and parietal bones were removed and the cerebellum was aspirated to expose the surface of the medulla
Recordmg Activity from single n e u r o n s was isolated by mlcroplpettes (l d = 1 0 Hm, Z = 5 - 1 0 M D ) filled with 1 6 M K citrate Initial coordinates used to locate the NTS were 2 7 m m anterior to the obex, 1 7 m m lateral to the midhne and 1 0 m m ventral to the surface of the medulla The electrode was advanced in 50 ktm steps and neural activity tested until robust gustatory responses were e n c o u n t e r e d T h e preparation was then left undisturbed for approximately 30 mln to permit the tissue to settle around the electrode This increased the probability of achieving the long-term stability necessary to maintain adequate isolation of single neurons over both the pre- and post-amdorlde stimulus series Action potentials were
identified by consistency ot wa'~eform spike a m p h t u d e and the requirement that no two spikes occur within an interval ot less than 1 5 ms Single-unit potentials were amplified filtered and dtsplaw.d on an oscilloscope using conventional recording techniques A direct-coupled 4-channel tape recorder was used to preterit, unit activity, the onset m a r k e r pulse and a voice commentar~ lot later analysis off-hne
Sttmuh and ~ttmulus dehvery A total of 15 stlmuh were employed (see Fable I) The emphasis in th~s array was on Na-Lx salts, but each of the other recognized basic tastes was represented, as were complex combinations of the basic tastes (e g Na saccharin) and stimuli that evoke unique taste perceptions (MSG, polycose) All solutions were prepared ,n dlstdled water except sucrose and glucose to which 5% tap water was added to assure adequate conductivity tot reliable triggering ol a stimulus onset marker Five milliliters of solution was sprayed over the tongue at a rate of 2 ml/s Gustatory-evoked activity was recorded for 5 s following stimulus onset The m o m e n t of stimulus contact with the tongue was marked by a T T L logic device which passed 11-nA current through the rat 9, an a m o u n t two orders of magnitude below the threshold for electric taste 8 2,~ Each tastant was followed by a 20-ml D H z O rinse and by a m i n i m u m rest period of 60 s Stimuli were presented at mtervals of no less than 90 s to prevent possible adaptation effects Additional rinses and rest periods were occasionally required for basehne activity levels to be reestabhshed Stimuli were presented m a quasi-random order with the stipulation that chemicals representing similar taste qualities not be applied consecutively If the neuron remained well isolated at the end of the stimulus series 180 ml of amflonde (0 5 m M ) was sprayed into the m o u t h at a rate of 1 ml/s for 3 m m This was followed b~ a 30-s rest and reapphcatlon ol the stimulus series D u n n g the post-amllonde period all procedures for stimulus application were identical to those used earher except that the amllorlde solution was used for rinses between stlmuh Data analysts D a t a were analyzed off-hne using an I B M - A T c o m p u t e r Spontaneous actwlty was subtracted from the evoked response to yield net spikes/s for the 5-s post-sUmulus period These counts were used to calculate response m a g m t u d e , breadth of t u m n g and average s p o n t a n e o u s rate for all cells prior to a n d following apphcatlon of amllorlde Derived analyses, including calculation of lnterst,mulus and interneuronal correlation matrices, m u l t l d l m e n s m n a l scahng, u m v a n a t e F-tests and post hoe comparisons were all performed with the Systat package s9 Hierarchical cluster analyses, used to Identify groups of cells and stimuli with similar functional properties, were conducted using the clustan routine on an I B M 3090 computer
Total
u-q Pre t~R Post
Spontaneous Rate
/
40
"-.
30
,.~ 10-
~.
20
r~
"~
tO
~ z
\
/
// 03
\
/ ~
-- Pre --- P o s t
/\
"~" 50
i',/
\ *
\\
~
/"--..
\!
f\
,, ,.
\
/*\
II
5. NINSN3N4
total
GI
G2
G3
G4
Fig 1 Mean s p o n t a n e o u s rate prior to and following amllorlde application for all neurons (total, n = 37) and for each of the 4 subgroups ( G 1 - G 4 ) All differences are non-significant
L M
K Ca Q Ct H
S
G $8 P
X
Fig 2 Mean response profiles across all n e u r o n s to the stimulus array prior to (dashed hne) and following (solid hne) lingual application of amflorlde Spontaneous activity h a s been subtracted from the total response to each stimulus Abbreviations are from Table I X mean response across all stimuli, * P ~ 0 05
249 Summary staustlcs mcludmg calculation of mean spontaneous rate, breadth of tuning and evoked rates were obtamed for each neuronal cluster as they had been for the entire population of cells Finally net spikes for each of 50 consecutwe 100-ms bins were averaged across umts to gtve post-sttmulus t~me Mstograms (PSTHs) for all sttmuh pre- and postamdonde From these, temporally based correlation matnces were calculated and mult~dtmens~onal spaces were generated based on the relative s~mtlartty of responses t~me courses
0 328 0 392 0 456 0 519
0 583 0 647 0711 0 775
RESULTS
0 838 0 902
General fmdmgs A c r o s s all cells r e c o r d e d in the NTS, stgnlficantly less acttvity was e v o k e d by sodium and lithium salts, M S G and sodium s a c c h a n n following amllorlde The magnitude of the decrease - - 47 1% to 0 1 M NaCI - - is consistent with that o b t a m e d from whole nerve chorda t y m p a n l responses in several species 7'i2J5'17-2°22 mmiloride did not affect the activity e v o k e d by sugars, acids or bitter stimuli M o r e revealing, however, was the effect of a m t l o n d e on subtypes of NTS taste cells Neurons could be categorized into 4 groups b a s e d on their response profiles to the entire stimulus array before the apphcation of amtlorlde (see division into neuron types below) G r o u p 1 (salt-sugar) showed g o o d sensitiwty to Na-Lt salts plus a side b a n d of responsiveness to the sugars G r o u p 2 (salt) neurons gave the same strong response to Na-Li salts, but were not effectively driven by any other stimulus G r o u p 3 (salt-acid) cells were equally sensitive to NaCI and LiCI but also r e s p o n d e d well to HCI and citric acids and to CaCI 2 G r o u p 4 (acid-salt-bitter) also r e s p o n d e d well to NaCI and LICI, but showed exquisite sensitivity to the two acids and a side b a n d of responstveness to the 3 bitter stimuli Thus, whtle the 4 groups showed roughly equal sensitivity to Na-LI salts, their full profiles p e r m i t t e d statistically reliable discrimination a m o n g t h e m T h e effect of amlloride was to suppress salt responses in G r o u p s 1 and 2 profoundly while leaving those in G r o u p s 3 and 4 unaffected
Spontaneous acttvtty and response cnterton The criterion for an e v o k e d response was a change in neural actlvlty of 1 65 S D ( P < 0 05, one-tailed) from m e a n s p o n t a n e o u s rate for each cell, sustained for 5 s M e a n s p o n t a n e o u s rate before the application of amiloride was 11 7 + 11 0 spikes/s, a value that did not differ significantly among the 4 subgroups (F3 32 = 0 429, non-significant) A m i l o n d e r e d u c e d the overall spontaneous activity by only a non-significant 1 3 splkes/s ( F 3 32
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= 0 837), no neuronal subgroup was slgntficantly suppressed, though G r o u p 2 (salt) n e u r o n s e x p e n e n c e d a reduction that a p p r o a c h e d significance (see Fig 1)
Evoked acttvlty Breadth of responstveness NTS cells showed typically b r o a d responsiveness across the sttmulus array p n o r to amiloride a p p h c a t i o n O f 555 total stimulus apphcatlons (37 neurons × 15 sttmuh) 78 9 % fulfilled the c n t e n o n for excitation, 0 4% for inh]bmon, and 20 7 % e v o k e d no response Following a m l l o n d e , t h e r e was a non-significant decline to 69 9% excitation, whtle 1 1% of the stimulus apphcations caused inhibition and 29 0 % no response Across all cells, the adminlstratton of amlloride did not result in sigmficant changes in b r e a d t h of responsiveness according to 3 s t a n d a r d measures the p r o p o r t i o n of stlmuh to which each n e u r o n r e s p o n d e d , the p r o p o r t i o n of neurons r e s p o n d i n g to each stimulus, or the breadth-of-tuning ( B O T ) metric *'36 H o w e v e r the n u m b e r of cells that r e s p o n d e d to Na-Li salts declined by 23 0% following amiloride The B O T metric p r o v e d m o r e r e v e a h n g when applied to the responses of neuronal subgroups T h e m e a n B O T was 0 79 pre- and 0 83 p o s t a m i l o r t d e (F] 32 = 3 796, n s ) But while most cells in G r o u p s 1, 3 and 4 showed marginal decreases in b r e a d t h due to the reduction of the sodium response c o m p o n e n t , G r o u p 2 (salt) neurons experienced a m a r k e d increase from 0 68 to 0 83 ( F 1,32 = 17 242, P < 0 001), a result that reflects their loss of responsiveness to the stimulus that c o n f e r r e d specificity
* The breadth-of-tuning metric prov2des an index of the dtstrlbutton of a cell's response across the 4 prototyplcal stlmuh It is calculated according to the formula H = -k~; p,logp, where H = breadth-of-tuning coefftcmnt, k = a scahng constant, whmh is 1 661 for 4 sttmuh, p, = the proportion of the neuron's response devoted to any one stimulus The value of H ranges from 0 00 for a neuron that responds to only one of the prototyplcal stlmuh, to 1 00 when the response ~s equal to all 4
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Fig 4 Mean response profiles of each subgroup of neurons prior to (dashed hne) and following (solid hne) amdonde apphcaUon Abbrevlauons are from Table I X, mean response to all stimuh, *, P < 0 05 (reprinted from Scott and Glza, Science, 249 (1990) 1585-1587 with permission Copyright 1990 by the AAAS)
General effect o f amdortde on response rate Activity e v o k e d by the 7 c o m p o u n d s containing Na ÷ or L1÷ d e c h n e d by 33 to 51% ( P < 0 003 in all cases) following the a p p h c a t l o n of a m t l o n d e , while responses to all o t h e r sttmuh were unaffected (Fig 2) W e a k e r NaCI solutions were inhibited p r o p o r t i o n a t e l y m o r e than those that were c o n c e n t r a t e d , and responses to the halide salts were s u p p r e s s e d m o r e than those of M S G and Na saccharin These effects are m o r e strIkmg when neural groups are defined and considered m d e p e n d e n t l y , as shown below Division mto neuron types T h e r e are many m o r p h o logical and functional criteria by which neurons may be c a t e g o n z e d 6 1 1 , 1 6 , 3 7 38 O u r interest m thts study was the influence of a m t l o n d e on taste-evoked activity Thus an a p p r o p r i a t e index for neuronal c a t e g o n z a t t o n is the response profile of a cell, both before amlloride apphcation to help organize the system in anticipation of the amtlortde, and afterward to d e t e r m i n e how taste cells are r e a h g n e d by its effects The net (total mmus spontaneous) discharges e v o k e d by each stimulus in 5 s constitutes the 15-point response profile for each of the 37 neurons before a m f l o n d e is a d m i n i s t e r e d Pearson p r o d u c t - m o m e n t c o r r e l a t m n coefflctents are then calculated between all pairs of profiles (n = 37 x 36 = 666 coefficients), providing a matrLx of relative functional slmIlanty among all cells This matrix may be subjected to a cluster analysis 4° the result of which takes the form of a d e n d r o g r a m (Fig 3) Neurons are n u m b e r e d in the o r d e r in which they were isolated Pairs of cells are Interconnected at the level of correlation between their response
profiles, groups are fused at the m e a n level of correlation between their constituent m e m b e r s in an l t e r a t w e process until all neurons in the sample are connected The suggestion from the p r e - a m t l o r l d e d e n d r o g r a m shown in Fig 3 is that our neural sample m a y be classified into 4 statistically dtstmct clusters that m a y be t r e a t e d independently m further analyses** B e n e a t h each cell n u m b e r in Fig 3 ts labeled the stimulus that e v o k e d the greatest response, followed by any o t h e r stimulus that elicited at least 80% of that m a x i m u m The left-hand cluster ( G r o u p 1) includes cells 1-37 (n = 9), whose p n m a r y sensitivity was to NaC1 (N)" but which also r e s p o n d e d relatively well to sugars and s o d i u m sacchann (S) This group includes response profiles that are fully mtercorrelated at a level of + 0 73 Next to it ts G r o u p 2 c o m p o s e d of cells 10-33 (n = 10) which r e s p o n d e d almost exclusively to NaCl and whose profiles are c o r r e l a t e d at a level of + 0 69 or a b o v e G r o u p 3 encompasses cells 4 - 2 6 (n = 8), all of which are also connected at a level of + 0 69 or a b o v e , this group ts characterized by nearly equal responses to NaCl and the acids G r o u p 4 includes cells 6 - 2 8 (n = 9), all lntercorrelated at + 0 71, and showmg p r e d o m i n a n t responsweness to the acids with strong sldebands to NaCl and to bitter substances In all 4 groups, the m e a n response to 0 1 M NaCl and LtCl was roughly equivalent at nearly 40 splkes/s Cell 17 had a profile that c o r r e l a t e d p o o r l y with all others, and so was excluded from the following analyses Effect o f armlonde by subgroup The application of
** We do not maintain that we have found exactly 4 gustatory neuron types The cluster analys~s also permits a stausncal d~wsion into just two groups (cells 1-17 and cells 4-28) or, conversely, into a large number of more exclusive clusters We take 4 to be a convenient and manageable number of rather clearly defined groups
251 amllorlde to the tongue had sharply contrasting effects on the responsiveness of neurons of different subgroups to Na-L1 compounds (Fig 4) The sensitivity of cells in Group 1 (salt-sugar) declined to the 4 concentrations of NaCl by a mean of 79%, to 0 1 M LIC1 by 74% and to 0 03 M Na saccharin by 30% (the response here was presumably sustained in part by the sweetness of saccharin in these sugar-sensitive cells) Their responses to all other stimuli were unaffected Group 2 (salt) cells showed a mean decline of 68% to the 4 concentrations of NaCl, 75% to 0 1 M LiCl, 58% to 0 1 M MSG and 52% to 0 03 M Na saccharin In contrast, the salt sensitivity of cells in Groups 3 and 4 was unaffected by amIloride Group 3 (salt-acid) neurons showed only an increase In response to citric acid, while Group 4 (acid-salt-bitter) cells experienced small but significant reductions to MSG and sucrose When the postamllonde response of each neuron to 0 1 M NaCl was expressed as a percentage of the pre-amIloride response, the difference among neuron groups was obvious (Fig 5) There was almost no overlap between the effect of amdoride on the first two versus the latter two groups, despite the fact that the mean pre-amdoride response to Na-LI salts was similar In all groups Th~s point Is dlustrated in Fig 6 which contains raw records of the responses of a representative cell from each group to 0 1 M NaCl before and after amilonde apphcat~on Thus, the broad response profile, rather than the response to Na-LI salts alone, provides nearly perfect predlctabdlty of which taste neurons in the central nervous system will be affected by interfenng with a transductlon mechamsm for Na and L1 ions Taste quahty Just as a 15-point profile across stimuli could be generated to represent the response pattern of each of the 37 neurons, so a 37-point profile across neurons may represent the response pattern evoked by each stimulus The correlation coeffioent between any pair of profiles is taken as an index of the relative similarity of the two taste qualmes they represent Alterations m the taste quality of a stimulus, occasioned here by the apphcatlon of amiloride, would appear as changes m the correlation between its profile and those of all other stimuh In Fig 7 we have plotted the correlaUons between the profile generated by 0 1 M NaC1 and those of the 14 other stimuli, both before (left column) and after amiloride Three distinct results emerge from this analysis First, 0 1 M NaCl largely maintained its close relationship with 0 1 M LICI, and the 3 remaining concentrations of NaC1, the mean of these 4 correlations being +0 92 pre- and +0 86 postamilorlde This minor reduction stems from the response suppression that typically yields lower coeffloents It is expected that these coeffioents should remain rather constant, for
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Fig 5 Response to 0 1 M NaCI following amflonde apphcatlon expressed as a percentage of each neuron's response before amdonde Valuesbelow zero indicate mhlbltory responses following amdonde Arrows mark the neurons whose mdwtdual responses are depicted m Fig 6 (GI, cell 21, G2, cell 11, G3, cell 9, G4, cell 21)
any influence of amllonde should be exerted across all 5 of the Na-LI salts we used Secondly, there was an increase In stmdanty between the profile of 0 1 M NaCl and those of the 5 sumuh that humans characterize as bitter-sour KCI, CaC12, quinine HC1, HCI and citric acid The mean of these correlations rose with amllonde application from +0 69 to +0 86, the same mean coefficient that 0 1 M NaCl had with other Na-Ll salts This result derives from the selective loss of responsiveness to Na-LI salts in neuronal Groups 1 and 2 The remaining activity evoked by NaCl and LICI is m Group 3 and 4 cells where sensitivity to bitter and sour stimuli is also high Thus there is increased covanance between the neurons that respond to Na-Ll salts and the bitter chemicals and acids, and so higher correlations between their profiles Finally the coefficient between 0 1 M NaCl and the unique tastes of MSG (meat-like) and polycose (starchy) was reduced moderately while its correlation with stimuli that humans describe as sweet - - sucrose, glucose, Na sacchann - - plunged from a mean of +0 68 to +0 26 The latter occurred because Group 1 neurons that shared a sensitivity to Na-LI salts and sugars, lost that common bond to the effects of amllorlde, and with it the covarlance that permitted at least moderate correlations Taken together, these results imply that the tastes of NaCl and LICI became predominantly sour-bitter followmg amiloride The matrix of correlations between all pairs of stimulus profiles (n = 15 x 1 4 - 2 = 105) may be used to create a mulUdimensional space In which the relative similarity of stimuli is proportional to their proximity Such a space,
252
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Fig 6 Responses of a representative neuron from each group to 0 I M NaCI prior to (left) and following (right) hngual apphcation of amdonde The evoked (total minus spontaneous) response declined by 92% both in cell 21 (GI) and m cell 11 (G2) while tt rose 1% m cell 9 (G3) and decreased by 6% m cell 13 (G4)
derived from neural responses before a m d o n d e apphcatlon, ~s shown m Fig 8 A In It may be seen a typical distribution of stimuli, with sugars and bitter-sour st~muh located at opposite extremes, Na-L1 salts and M S G form a tight central group, while polycose is d,stmct from all o t h e r sUmuh The space m Fig 8B is g e n e r a t e d from responses r e c o r d e d after a m d o n d e t r e a t m e n t As exp e c t e d from the preceding analysis, the 5 Na-L] salts have m o v e d to the sour-b,tter extreme, leaving only M S G m
the p o s m o n they formerly occupied It seems a reasonable mference that if the Na-LI salts r e p r e s e n t the perception of 'saltiness' m the n o r m a l l y funcUonmg taste system (Fig 8A) then this quahty has been lost with a m d o n d e (Fig 8B) T h e r e f o r e , with the Na-Ll response suppressed m G r o u p 1 and 2 neurons, 'saltiness' a p p e a r s to be e h m m a t e d although a p p r o x i m a t e l y 50% o f the total activity e v o k e d by these salts remains 'Saltiness' may be coded by neurons whose profiles emphasize Na-L! re-
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sponsweness and exclude sensmvaty to other non-sweet stamuh These cells may be served by receptors that use
a passive transport channel (susceptable to amflonde blockade) as a transductaon mechamsm, as opposed to receptors lnnervatmg those neurons that respond broadly to non-sweet chemicals, including NaCI and LICI These latter neurons may be responding to the halogen anaon of the NaCI molecule 15 Temporal analysts The correlataons among stamulus profiles above are based on the total response of each cell to every sUmulus, summed over the 5-s post-stamulus recordmg period Thus the tame course over whach the response develops is not considered To analyze temporal propertaes, each response was dwaded into fifty 100-ms bans and summed across neurons for a gwen stamulus Thas represents the temporal response profile for that chemacal Correlataons among all pmrs of profiles (n = 15 x 1 4 - 2 = 105) could be calculated and multadlmensaonal spaces - - now based on temporal propertaes - - generated as before Spaces derwed from responses before (Fig 9A) and after (Fag 9B) amdonde apphcatlon are remarkably samdar, and mdacate that the severe suppressaon of Na-La responses occurs m a temporally uniform manner In both spaces, stamuh that are characterized by hagh phasac and relatwely low tomc actwaty are posatloned toward the left, while those with low phasic-
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Fig 8 Three-dimensional space representing the relative s,mdarmes of response profiles evoked by each stimulus before (A) and after (B) amdorlde Abbreviations are from Table I The axes are not defined (reprinted from Scott and Giza, Scwnce, 249 (1990) 1585-1587, with permtsseon Copyright 1990 by the AAAS)
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254 to-tomc ratios are s~tuated at the right DISCUSSION
Imphcattons for gustatory codmg The most striking findings of this study of NTS taste cells are (1) the effects of a m d o r l d e on responsweness to NaCI and LIC1 are either profound or minimal, d e p e n d mg on the cell, and (2) the response profile of any neuron permits a confident predtct~on of which result wdl be o b t a i n e d for that cell In G r o u p s 1 and 2, 15 of 19 cells had their Na-L1 responsweness suppressed by m o r e than 7 5 % , m G r o u p s 3 and 4, 15 of 17 were suppressed by less than 25% This provides clear evidence that r e c e p t o r cells using a passwe s o d m m transduction mechanism relay through p e r i p h e r a l nerves to ~dentffmble subgroups of CNS taste cells M o r e o v e r , when this mechamsm is b l o c k e d by a m d o n d e , the r e m a m m g actw~ty e v o k e d by Na-L1 salts is highly slmdar to that e l i o t e d by a o d s and b~tter c o m p o u n d s This ~mphes that the actw~ty of G r o u p 1 and 2 neurons ~s responsible for the p e r c e w e d distraction between 'saltiness' and sour-bitter taste and so argues for the notion that certain taste cells perform specific coding functions A m l l o n d e has been r e p o r t e d to cause an increase m m e m b r a n e resistance and a hyperpolanzat~on of 20 mV m a subset of rat taste r e c e p t o r cells 2 3 These receptors, which constitute about 25% of the population, should subsequently be responsive only to st~muh that cause s u f f t o e n t d e p o l a r i z a t i o n to o v e r c o m e this suppresswe influence The i n t e n s i t y - r e s p o n s e functions we r e c o r d e d from G r o u p 1 and 2 cells m the NTS across 4 concentrations of NaC1 fulfill thts expectation p o s t a m d o n d e response functions rise monotonically with stimulus intensity, though actwlty levels are suppressed some 75% from those r e c o r d e d m the u n a d u l t e r a t e d system*** F u r t h e r m o r e , ff a r e c e p t o r cell is h y p e r p o l a n z e d by a m d o r l d e its responsiveness to all stlmuh should be suppressed, regardless of the mechanism by which it is n o r m a l l y d e p o l a r i z e d Thus the actwlty aroused m that cell by sugars, a o d s , bitter salts and alkaloids should be r e d u c e d as much as that to NaCI and LICI The fact that responses m the NTS to all stimuli that did not contain Na or Ll ions were unaffected by a m d o n d e apphcatlon ~mphes that the transductlon of these sUmuh ~s accomphshed entirely by r e c e p t o r cells that are not a m d o n d e sensitwe Thus there ~s a sharp distmct~on, estabhshed at the r e c e p t o r level and m a i n t a i n e d in peripheral nerves to be manifested in the m e d u l l a amdorlde-sensltwe receptors recognize the presence of Na + and L~+ but do not
part~opate m the transduct~on of any o t h e r class ot tast~ stimulus These receptors project p r e d o m i n a n t l y to neurons we have included m G r o u p s t and 2 which are thus r e n d e r e d h y p o r e s p o n s w e by a m l l o n d e G r o u p 1 neurons presumably receive input as well from a separate subset of receptors that transduce sugars, and these responses remain following a m d o n d e t r e a t m e n t G r o u p 2 cells are largely deprived of their only m a j o r source of lnnervatlon and may r e s p o n d just to residual activity from sodmm receptors and perhaps to m i n o r mputs trom others Thus we are led to conclude that coding channels exist within the taste system, a p o s m o n that is d e v e l o p e d more tully elsewhere ~
The taste quahty of NaCl Despite the increase m simllartty b e t w e e n Na-Ll salts and stlmuh that humans label sour or bitter, fireddeprived rats are r e p o r t e d to accept NaCI m o r e wdhngly after their tongues are treated with a m d o n d e 5 lo This would imply an i m p r o v e m e n t m h e d o m c quahty, m contrast to the tmpllcatlons of o u r electrophystologlcal results H o w e v e r , this behavtor ts only seen when NaCI ~s p r e s e n t e d at concentrations of 0 3 and 0 5 M The aversweness of these stimuli would be a t t e n u a t e d by amllorlde-lnduced suppresston, such that the net result of changes m p e r c e w e d intensity (toward m o r e acceptable levels) and quahty ( p r e s u m a b l y t o w a r d those that are less acceptable) are unpredtctable I n c r e a s e d acceptance does not result from a m l l o n d e t r e a t m e n t when the stimulus is a m o r e palatable 0 1 M NaCI Thus, there is r o o m within the bounds of our data to a c c o m m o d a t e the behavioral findings In support of the notion of a shift in quahty, Bernstem reports that s o d m m - d e p r i v e d rats, whose physiological need renders 0 5 M s o d m m p a l a t a b l e , are less wdhng to consume th~s solution when they are p r e t r e a t e d w~th a m d o r t d e T h e r e f o r e , when intensity ~s not a deterrant, the p r e s u m e d shift of the quahty ehc~ted by NaCI t o w a r d m o r e hedomcally n e g a t w e tastes is behaviorally manifest
The tastes of MSG and polycose A m l l o r t d e suppressed responses to M S G by httle more than half as much as those to NaCI and L1CI H o w e v e r , the taste quahty of M S G r e m a i n e d firm, even as those of NaCI and L~C1 m o v e d t o w a r d bitter-sour c o m p o u n d s (Fig 8) This is quite slmtlar to the effect on Na saccharin, whose responses were suppressed to the same degree as those of M S G and which also m a i n t a i n e d its p o s m o n m the stimulus space after a m d o r i d e Just as Na saccharin has a d o m i n a n t quahty (sweet) to sustain Its
*** It is puzzhng, however, that spontaneous actlvuy in these same cells, which might be expected to be most suppressed by a severe hyperpolanzatlon of the receptors that serve them, was reduced by only 2% (Group l) and 35% (Group 2)
255 l d e n t a y when saltmess has been blocked, so presumably
taste ~s w h e t h e r the afferent signal ~s read across the
does M S G ( U m a m 0 41 Thus these data provide evidence
entire population of n eu r o n s or ~s restricted to a channel
for a taste quahty for M S G that transcends saltiness, and
of activity most sahent to the transmission of a particular
which ~s not transduced p n m a r d y through passive s o dm m
quahty
channels
nism through a m d o n d e apphcat~on has r e v e a l e d deficits
Polycose has also b e e n touted as the p r o to ty p e for the i n d e p e n d e n t taste quahty 'starch '32 In support of th~s,
N TS that p r o m o t e the latter v~ew
T h e d~sabhng of a s p e o f i c transduct~on mecha-
m the responses of ~denUfiable neural subgroups m the
polycose ehc~ted an actlwty profile that was poorly correlated w~th those of all accepted prototypes and which was unaffected by amllortde
Conclusion A m a j o r ~ssue m d~scuss~ons of the neural code for
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