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Differential arousal response to gustatory stimuli in the awake rabbit
Electroencephalography and Clinical Neurophysiology, 1979, 47 : 1--11
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© Elsevier/North-Holland Scientific Publishers, Ltd.
DIFFERENTIAL AROUSAL RESPONSE TO GUSTATORY STIMULI IN THE AWAKE RABBIT J.E. STEINER and J. REUVENI
Department o f Oral Biology, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem (Israel) (Accepted for publication: October 19, 1978)
The perception of gustatory stimuli may be measured behaviorally or electrophysiologically. Although the sensory modality of taste may be subdivided into a relatively small number of quality categories, another split is apparent for these sub-modalities, namely the hedonic dimension, reflected behaviorally by indifference, acceptance or rejection (Pfaffmann et al. 1977). As contrasted with other sensory modalities only little is known about cortical taste mechanisms and their electrophysiological correlates. Information is mainly related to attempts to record single cell activity or evoked responses {Cohen et al. 1971; Funakoshi and Kawamura 1971; Sudakov et al. 1971; Funakoshi et al. 1972; Yamamoto and Kawamura 1972, 1975). A definitive statement of EEG correlates of gustation remains to be made. In the present experiments an ECoG study was undertaken to compare the arousal response induced by the intraoral presentation of deionized water, with those induced by identical presentation of several concentrations each of sweet, salty, sour and bitter stimuli. The unanesthetized and restrained rabbit was chosen for these experiments because: (a) the electrical brain activity of this animal is well known in different physiological and pharmacological conditions (Monnier and Gangloff 1961; Longo 1962); (b) the behavioral response of this species to tastants has been studied {Carpenter 1956; Ganchrow in preparation).
Methods
Records were obtained from 17 'chronic' preparations of male rabbits (Lepus europaeus) (2.6 kg average body weight}. Epidural silver screw recording electrodes were implanted according to the widely accepted stereotaxic method of Monnier and Gangloff {1961), under pentobarbital anesthesia (50 mg/kg, b.w.) and local anesthesia of the head and eye region (Lignocalne 1% solution infiltration). In all experiments the right eye was enucleated to permit free access to the region of the rhinal sulcus. Electrodes were implanted in the orbital wall and in the region of the limbic (A, B) and occipital cortex (C) as shown in Fig. 1 according to stereotaxic coordinates. In 7 of the animals the brain was later removed and the location of the electrodes verified. After surgery animals received appropriate local and systemic antibiotic treatment. Rapid recovery was confirmed by normal motor behavior as well as regular food and water intake. Taste experiments were carried out about 3--6 days after surgery in a shielded and air-conditioned laboratory. For the actual experiment the animal was placed in a special sling (Monnier and Gangioff 1961) which minimized body movements. No pharmacological agents were given. The sapid substances and their concentrations are presented in Table I. All chemicals were reagent grade and were dissolved in deionized water. For sour stimuli two different chemicals were chosen, acetic acid and ascorbic acid. The
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J.E. S T E I N E R , J. R E U V E N I B
C
D
E
F
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0 3 6
A
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15 m Ttmm
SUTURA SAGITTALIS c
b
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b
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latter was selected in order t o sample a sour stimulus n o t causing simultaneous olfactory or trigeminal stimulation, as acetic acid m a y do. Taste stimuli as well as deionized water were delivered to the dorsal surface o f the tongue with the m o u t h closed, f r om outside the Faraday cage via a gravitational flow system for a fixed duration o f 5 sec and a fixed 10 ml volume. Water rinses lasted 20-25 sec and repose between stimuli at least 10 min. All stimuli were at r o o m t e m p e r a t u r e (23°C) and presented in a r andom i z e d sequence. In o r d er to diminish reflex swallowing during stimulus flow, the h a m m o c k was tilted slightly d o w n w a r d at t he head end, facilitating drooling o f the fluids o u t of the
m out h. Records were perform ed with a 12channel apparatus. The brain activity was simultaneously recorded on a 4-channel magnetic tape recorder. The arousal response appearing after water and sapid stimuli were evaluated according to the following 3 parameters: (a) Duration of arousal response was measured in seconds, from the onset o f the stimulus for as long as arousal was detectable by visual inspection o f the tracing. (b ) Change in frequency was determined by a modified technique based on Sulg (1969) for manual analysis o f frequencies. The resting trace was sampled during a 6 sec period prior to stimulus presentation. The frequency in this sample was c o m p a r e d with t hat for the 6 sec period starting from the 'stimulus off' mark of the actual presentation. The prestimulus frequency was arbitrarily labeled as 100% and the change was expressed as percentage increase. (c) Changes in amplitude were quantified from the brain activity recorded on magnetic tape. Tapes were analyzed by a POP-15/140 computer. The absolute average amplitude was determined for a 6 s e c prestimulus period. This value was again labeled arbitrarily as 100%. The absolute average amplitude o f a 6 sec poststimulus period was expressed as a percentage of the prestimulus amplitude. From the individual measures calculated in each o f the stimulus trials for each experimental animal and across stimulus types (tastes) mean values were calculated. The means obtained in those trials across all animals, where water was presented as stimulus (n = 60), served as control value. The duration of the arousal, the change in frequency and the change in amplitude induced by the tastants were com pared to the values obtained after water (control) stimulation. Furthermore, the means, across all trials used in each o f the p a r t i c u l a r stimulus concentrations, were also com pared in order to assess a doseresponse relationship. A c o m p u t e r i z e d program (ANOVA) for a two-way analysis of variance was used to
D I F F E R E N T I A L A R O U S A L TO G U S T A T O R Y STIMULI
assess the significance of the differences found between control and tastant stimulations as well as between the values found with different stimulus concentrations (Kim and K o h o u t 1976).
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Deionized water was presented to all animals, with a total of 60 trials. This stimulus was found to induce an arousal reaction which lasted 14.0 (+ 2.3) sec; it caused a 4.7% increase of the ECoG frequency and a 25% amplitude reduction as compared to the prestimulus conditions. The values thus obtained are presented as control values in Tables II-VI.
across the different concentrations (Table III). It can be seen that t w o of the salt concentrations, that o f 1.0 and 2.0 M, induced an arousal significantly different from that induced by water, both in terms of duration and of amplitude depression. Comparing the arousal reactions induced by different NaC1 concentrations, it was found that increasing the concentration significantly prolonged the duration of the arousal. Comparing the frequency increment caused by increased NaC1 concentrations, one can see that the lowest salt concentration used, 0.1 M, produced an increment similar to that of water. But 0.5 M, 1.0 M and 2.0 M salt solutions induced markedly higher frequency increases than water or the weakest NaC1 solution did.
(b) Sweet
(d) Sour
Sucrose was used in concentrations listed in Table I in 14 experiments with a total of 57 trials across the different concentrations (Table II). It can be seen that the arousal reaction in response to different sucrose concentrations showed only a slightly significant difference from the reaction caused by deionized water ('tasteless' or control) stimulation.
Results were obtained for ascorbic acid in 11 experiments with a total o f 40 trials (Table IV). From this table it may be seen that stimulation with ascorbic acid induced a markedly different arousal from that caused by water. All b u t the weakest (0.01 M) concentration induced a significantly longer lasting arousal than did water. Frequency increments were observed for all but the weakest concentrations. The differences between the waterinduced frequency increment and that caused by the t w o highest ascorbic acid concentrations (0.3; 0.5 M) were found to be highly sig-
Results
(a ) Tasteless
(c) Salty NaC1 was used in concentrations listed in Table I to present salty stimuli to the animals, in 19 experiments with a total of 64 trials
TABLE I Stimulants and their concentrations (in M) presented intraorally. Modality :
Sweet
Salty
Sour
Stimulant:
Sucrose
NaCI
Aseorbic acid
Acetic acid
Quinine HC!
0.1 0.5 1.0 2.0
0.1 0.5 1.0 2.0
0.01 0.1 0.3 0.5
0.025 0.05 0.125
0.001 0.01 0.03 0.05
Bitter
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J.E. S T E I N E R , J. R E U V E N I
T A B L E II Arousal r e s p o n s e to sucrose s t i m u l a t i o n . Parameters
R e s p o n s e d u r a t i o n in sec Mean S.D. F r e q u e n c y i n c r e m e n t in % (prestim. freq. = 100%) A m p l i t u d e d e c r e m e n t in % (prestim. amplit. = 1 0 0 % )
H20
14.0 (2.3) +4.7 --25
S t i m u l a n t c o n c e n t r a t i o n in M
Significance o f F in A N O V A test *
0.1
0.5
1.0
2.0
16.6 (3.2)
20.8 (4.4)
18.0 (3.3)
21.3 (2.3)
+10.2 --11
+4.6 --17
+13.9
+18.0
--28
--26
0.1--2
0.02
0.5--2
0.04
* Signs a n d n u m b e r s o n t h e left side o f this c o l u m n s h o w t h e pairs o f s t i m u l i c o m p a r e d . R i g h t side n u m b e r refers to level o f significance.
nificant. Furthermore, a gradual doseresponse relationship was apparent. The depression of amplitude also showed a similar trend of gradual effect. In 5 of the relevant comparisons, differences were found to be significant. Acetic acid was used in 9 experiments with a total of 37 trials {Table V). As Table V makes clear, acetic acid also induced a markedly different arousal from that caused
by water stimulation. The increasing concentrations of the stimulant resulted in a gradually prolonged duration of the arousal, as well as in an increment of frequencies. A similar trend could be observed for amplitude depression. The 0.125 M concentration of acetic acid was found to reduce the amplitude of the brain activity more markedly than did any other stimulant used in this experiment.
T A B L E III Arousal r e s p o n s e to NaCI s t i m u l a t i o n . Parameters
R e s p o n s e d u r a t i o n in sec Mean S.D.
F r e q u e n c y i n c r e m e n t in % (prestim. freq. = 100%) A m p l i t u d e d e c r e m e n t in % (prestim. amplit. = 100%)
H20
S t i m u l a n t c o n c e n t r a t i o n in M 0.1
0.5
1.0
2.0
14.0 (2.3)
13.0 (4.2)
19.1 (4.4)
19.7 (4.0)
34.8 (5.7)
+4.7
+2.2
--25
--30
+17.4
--28
+7.3
--34
Significance o f F in A N O V A test * H20--2 H20--1 0.1--2 0.5--2 0.1--0.5 l --2
0.002 0.001 0.001 0.03 0.02 0.007
+22.6
0.1--2
0.03
--43
H20--2 H20--1
0.05 0.05
* Signs a n d n u m b e r s o n t h e left side o f this c o l u m n s h o w t h e pairs o f stimuli c o m p a r e d . T h e r i g h t side n u m b e r refers t o t h e level o f significance.
D I F F E R E N T I A L A R O U S A L TO G U S T A T O R Y STIMULI TABLE IV Arousal response to ascorbic acid stimulation. Parameters
Response duration in sec Mean S.D.
Frequency increment in % (prestim. freq. = 100%) Amplitude decrement in % (prestim. amplit. = 100%)
H20
Stimulant concentration in M 0.01
0.1
14.0 (2.3)
11.44 (3.22)
19.28 (4.29)
37.22 (12.98)
61.23 (15.8)
+4.7
+3.0
+9.1
+19.7
--43
--23
--24
--23
0.3
0.5
Significance of F in ANOVA test * H20-0.5 H20--0.3 H20--0.1 0.01-0.5 0.01--0.3 0.1 - 0 . 5 0.1 - 0 . 3
0.001 0.001 0.03 0.001 0.001 0.002 0.03
+29.5
H20-0.5 H20-0.3 0.01-0.5
0.01 0.01 0.01
--47
H20-0.5 0.01-0.5 0.01-0.3 0.1 - 0 . 5 0.1 - 0 . 3
0.01 0.005 0.01 0.006 0.03
* Signs and numbers on the left side of this column show the pairs o f stimuli compared. The right side number refers to the level of significance.
(e) Bitter Quinine hydrochloride was used in concentrations shown in Table I to present animals with bitter stimuli, in 13 experiments with a
total of 47 trials. From the data presented in Table VI one can see that the bitter stimulant induced a clearly different arousal reaction from t h a t to water. For all 3 measured param-
TABLE V Arousal response to acetic acid stimulation.
Parameters
Response duration in sec Mean S.D.
Frequency increment in % (prestim. freq. = 100%) Amplitude decrement in % (prestim. amplit. = 100%)
H20
14.0 (2.3)
+4.7
--25
Stimulant concentration in M 0.025 (1%)
0.05 (2%)
0.125 (5%)
25.0 (7.4)
28.4 (6.8)
83.1 (17.4)
Significance o f F in ANOVA test *
H20--0.125 H20--0.05 H20--0.025 0.025--0.125 0.05 --0.125
0.001 0.001 0.001 0.003 0.001
+18.6
+24.8
+27.0
H20--0.125 H20--0.05
0.01 0.01
--37
--46
--51
H20--0.125 H20--0.05
0.001 0.006
* Signs and numbers on the left side of this column show the pairs of stimuli compared. The right side number refers to the level o f significance.
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J.E. STEINER, J. R E U V E N I
TABLE VI Arousal response to quinine stimulation. H20
Parameters
Response duration in sec Mean
S.D.
F r e q u e n c y increment in %
Stimulant concentration in M 0.001
0.01
0.03
0.05
14.0 (2.3)
20.3 (5.4)
22.7 (7.8)
27.6 (6.3)
39.8 (11.8)
+4.7
+5.9
--25
--16
H20--0.05 H20--0.03 0.001--0.05 0.01 --0.05 0.001--0.03
0.001 0.001 0.006 0.02 0.05
+17.5
+28.8
+40.0
H20--0.05 H20--0.03 0.001--0.05 0.01 --0.05 0.03 --0.05
0.01 0.03 0.001 0.002 0.03
--25
--33
--44
H20-0.05 H20--0.03 0.001--0.05 0.001--0.03 0.01 --0.05
0.007 0.04 0.002 0.02 0.01
(prestim. freq. = 100%)
Amplitude decrement in % (prestim. amplit. = 100%)
Significance of F in ANOVA test *
* Signs and numbers o n the left side o f this c o l u m n s h o w the pairs of stimuli compared. The right side number refers to the level of significance.
eters there was a clear gradual effect as the concentration of the stimulant increased. Data related to stimulation with 0.03 and 0.05 M concentration o f quinine differ significantly from those related to the water stimulation. Furthermore, most comparisons
between the results obtained by increasing stimulus concentrations also reveal significant differences. To gain an overview, the measures of the responses to the lowest and highest concentrations of each stimulant are compared with
E.E.G. AROUSAL UESPORSEIRTHEAWAKE RAB811 TO THE LOWEST AND HIGHEST CONCERTRATIOHSOF TASTANTS Tastants: H20 SUCROSE
001
--1
concentration
Highest
concentration
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:=
zo
28
Lowest
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ASCORBIC ACID o~o
ACET,C AC,D ~
l I
0
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,
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0.5
AMPLITIJBE RATIO POST-STIMI PRE-STIM
;
P I0
0
50 100 0 1 30 0 50 RESPOIfSE TIME (see) FREO,UEN£Y INCREASE{'/.) [RELATE{] TOSPONTANEOUSA['TIWTY}
Fig. 2. Comparative bar-graph of the values obtained in all 3 parameters used for the quantification of the arousal response. Concentrations ot tastants in M .
DIFFERENTIAL AROUSAL TO GUSTATORY STIMULI measures of water response in Fig. 2. It shows that: (a) Duration o f the arousal reaction induced by the lowest concentration of all sapid substances given in this experiment did n o t differ significantly from that induced by water (tasteless). The highest concentrations of the tastants, however, elicited a markedly longer lasting arousal than did water. Except for sucrose, this difference was found to be significant, the longest lasting arousal was observed in response to stimulation with 0 . 1 2 5 M acetic acid. (b) The increment in frequency caused b y the lowest concentrations of the tastants applied did n o t reveal any difference from that induced by water, with the exception of acetic acid in 0.025 M concentration, b u t even this was n o t significant. By contrast, the highest stimulus concentrations all caused a marked frequency increase as compared to water. Responses to bitter and sour stimuli differed significantly, while sweet and salty responses were only tending towards significance. 0.05 M quinine hydrochloride induced the highest frequency increment. (c) The decrement in amplitude caused by the lowest concentrations of the stimulating c o m p o u n d s was similar to that induced b y water. Only the lowest acetic acid concentration induced a slightly more detectable b u t still not significant amplitude decrem e n t as compared to the water effect. The highest concentrations of the salty, sour and bitter stimulants induced more marked amplitude decrements than did water or sucrose. 0.125 M acetic acid was found to have the most effect.
Discussion Findings presented here demonstrate that intraoral taste stimulation o f the adult restrained but non-anesthetized rabbit induced EEG arousal. The observed arousal response was f o u n d to be different when water was used as stimulant from that which appeared following taste stimulation. In response to sour and bitter tasting substances
7 the arousal showed a dose-dependent gradual increase, as indicated by 3 different quantifying parameters. These dose-dependent effects were expressed to a lesser degree with salty stimuli and were found n o t to be significant with sucrose. Ranking the effectiveness of sapid stimuli in inducing ECoG arousal, differentiable from that induced by water, gave the following order: quinine > acetic acid > ascorbic acid > NaC1 > sucrose. The results of the present study may be interpreted as reflecting in a quantitative manner (encephalographieally) the hedonic aspect of the tasting experience. In psychophysical testing situation, hedonics may be rated using the t w o bottle preference test. In this procedure the relative liquid consumption is used to assess taste preference (acceptance and rejection). In this testing situation it can easily be determined whether a certain tastant is perceived different from water in a positive or negative way. Comparing the data from behavioral testing (two bottle preference test} with the ECoG parameters reported here, one can conclude that tastants and their concentrations which were found aversive in the behavioral test, produced a more marked ECoG arousal than those which were preferred. Rabbits clearly discriminate between water and sucrose at 0.01 M concentration. A definite preference toward this tastant was observed at 0.1 M concentration. At the concentration of 1.0 M the animals again equally consumed sucrose and water while concentrations higher than 1.0 M were clearly rejected (Carpenter 1956). From the results of the present study it can be seen that 0.5 M sucrose markedly enhanced the duration o f the arousal as compared to the water response. Those concentrations, which were found in the mentioned behavioral study to be rejected, led to a certain increase in frequency. Although these differences were n o t found to be significant, y e t the ECoGs indicate an increase, although not a dramatic one, in vigilance and awareness, presumably reflecting aversion or dislike. It
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seems that the dislike of the animals for the above-optimal concentrations of sucrose is more clearly measurable in the behavioral response than from the parameters of EEG arousal. Salty, sour and bitter stimulants in certain concentrations were found to be rejected by the rabbit in the psychophysical testing situation (Carpenter 1956; Ganchrow, submitted for publication). The ECoG data obtained in this study reveal marked alertness and arousal towards that particular range of concentrations which was found to elicit behavioral rejection in this species. From behavioral data it is known that rabbits discriminate between water and 0.01 M NaC1. Preference was observed around 0.1 M. A clear rejection was observed at 0.5 M. The behaviorally preferred salt concentrations caused an arousal reaction which was n o t significantly different from the water response, while the arousal induced by NaC1 in concentrations above 0.5 M produced a significantly different arousal from that induced b y water. The unpleasantness of the 0.5, 1.0, and 2.0 M salt solutions is reflected b y the duration of the response, as well as by frequency increment and amplitude decrement. One may conclude that the unpleasantness of the salty taste causes a marked b u t still not drastic arousal effect. Behavioral data indicated that rabbits did not differentiate ascorbic acid from water at concentrations o f 0.001 M and 0.01 M, while they clearly rejected this c o m p o u n d in 0.02 and 0.05 M concentrations (Ganchrow, manuscript submitted). Comparing the behavioral and arousal data, it can be stated that the behaviorally rejected concentrations of ascorbic acid induced indeed an arousal which differed significantly from the water response. Thus once again intensity of the arousal may be interpreted as an objective sign of tasteaversion. Acetic acid was found n o t to be discriminated from water b y rabbits in concentration of 0.0001 M. A clear behavioral rejection was observed in 0.001 and in 0.01 M concentra-
J.E. STEINER, J. REUVENI
tion (Ganchrow, manuscript submitted). The arousal data evidenced very markedly and significantly different responses from the waterinduced arousal, with 0.025 M up to 0.125 M concentration of this c o m p o u n d . The intensity of the arousal response was found dosedependent. One can, therefore, consider these parameters as an objective indication of taste aversion. Quinine was found in the present experiment to be the most effective sapid stimulant producing an arousal response. Behavioral data (Carpenter 1956) showed clear rejection of this c o m p o u n d in concentrations above 0.0001 M. The dose-dependent EEG findings obtained in the present study strongly support the assumption that the marked arousal, highly significantly different from the water response toward increasing concentrations of this substance, is indicative of taste aversion. Searching the relevant literature, no experimental study showing similar results could be found regarding any mammalian species. Yet, in one avian species, namely the chick (Gallus domesticus L.) an effort was made to use the length (duration) of EEG arousal to indicate objectively behavioral preference and rejection (Gentle 1972). It was found that many of the solutions which did n o t induce any behavioral response in the birds gave no EEG indications. Some acids were found to produce EEG reactions differentiable from those caused by water, even without concomitant behavioral responses. It was concluded that the duration of the EEG response in the chick may well be considered as a more sensitive detection technique of taste rejection than behavioral testing. As to the other animal experiments attempting to define a correlation between behavioral and electrophysiological data, it is perhaps worthwhile to note that a comparison of the firing rates of primary and secondary taste neurons with behavioral acceptance and aversion of salty taste in the rat, did not reveal any typical change in firing rate which might correspond to behavioral aversion (Pfaffmann et al. 1961).
D I F F E R E N T I A L A R O U S A L TO G U S T A T O R Y STIMULI
Most recently a study was reported on taste-induced activity of cells in the amygdala of the awake 'behaving' rabbit (Schwartzbaum and Morse 1978). In this experiment cells were found in n. centralis region of the amygdala to receive taste information. Analysis of the response pattern revealed evidence of units responding exclusively or highly differentially to tastants, presented as intraoral stimuli. These findings strongly suggest that it is possible to find, at the cellular level, physiological correlates indicating qualitative and motivational (hedonic) differences between different sapid stimuli. To date no study has been reported correlating EEG parameters to hedonic ratings b y humans. However, a summated cortical evoked response in man, due to intraoral water and sapid stimulation, was described (Funakoshi and Kawamura 1971) as being of two components. The first reflected water and apparently originated from tactile stimulation of the oral cavity; the second, more delayed, c o m p o n e n t o f the response was interpreted by these authors as strongly suggestive of reflecting the taste aspect of the intraoral stimulus. This part of the evoked response was also f o u n d to be concentrationdependent. In conclusion: it is perhaps n o t unjustified to assume that careful analysis o f tasteinduced EEG changes m a y become an objective indicator for both the relative intensity and hedonic dimension o f a perceived taste sensation. The findings of the present study strongly suggest such a possibility. One can also assume that the aversiveness of the unpleasant stimulus causes a more easily detectable reaction than pleasant stimuli may cause. In this c o n t e x t it is appropriate to mention that oro-facial m o t o r reactions of innate and reflex-like character were demonstrated in response to tastants as well as to some food-related odorants in the neonate human infant (Steiner 1973, 1974, 1977). Similar reactions were later also f o u n d in animals (Grill 1978; Ganchrow et al. manuscript submitted). These observations showed
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that sweet stimuli and pleasant food odors induced more delicate and moderate responses, whereas sour and bitter tastants and offensive food odors always elicited quick dramatic and massive muscle reactions. It was concluded from these studies that pleasant and agreeable chemical stimuli do n o t carry any message o f urgency, while to the unpleasantness of sour and bitter stimuli and to the offensive f o o d odor the aspect of alarming urgency is attached. The negative hedonic aspect of such taste and odor stimuli is then evaluated by the central nervous system as an alarming sign, activating neural mechanisms of avoidance, defense and withdrawal. One may, therefore, state that just as offensive taste and smell stimuli activate more dramatic m o t o r reflexes than pleasant ones, so aversive tastants induce more drastic EEG signs of awareness and vigilance than indifferent or pleasant sapid materials do. In order to reach conclusive results, further experimentation is necessary using both different species of animals and human examinees. In such experimental conditions a correlation might be established between objectively detectable EEG signs and psychophysical rating and ranking of relative intensity and hedonics of perceived taste sensations.
Summary ECoG arousal response as elicited by deionized water and by several concentrations of sweet, salty, sour and bitter tasting substances, all applied intraorally, were studied in the awake restrained rabbit. The study was carried o u t on 17 chronic preparations o f adult male animals (2.6 kg average b o d y weight). Water as a stimulant was presented in 60 trials and tastants across qualities and concentrations in 245 trials. Arousal was quantitatively characterized b y its duration, frequency increment and amplitude decrement as compared to prestimulus conditions. Arousal induced b y tastants was
10
compared to that induced by water. Water and tastants induced arousals differentiable by the parameters measured. Further, most tastants produced a dose-dependent response. Comparison of ECoG and behavioral data clearly indicate that tastants inducing behavioral aversion also produce an arousal which significantly differs from response to water while behavioral preference was found not to show similar correspondence with ECoG data. The possibility of using arousal as an objective indicator for taste aversion is discussed.
R~sum~ Activations ECoG spdcifiques aux stimulus gustatifs chez le Lapin dveilld On a ~tudi~, chez le Lapin ~veill~ mais en contention, les activations ECoG suscit~es soit par de l'eau d~sionis~e, soit par des solutions en diverses concentrations, de substances sucr~es, salves, acides et am~res, toutes appliqu~es dans la cavit~ buccale. L'analyse a port~ sur 17 animaux, m~les adultes, en preparation 'chronique' (poids m o y e n 2,6 kg). L'eau a ~t~ utilis~e en tant que stimulant au cours de 60 essais et les diff~rentes substances gustatives dans 245 essais. La r~action d"arousal' a ~t~ quantitativement appreci~e par la dur~e, par l'augmentation en fr~quence et la diminution d'amplitude qui l'accompagnait, et compar~e l'activit~ pr~stimulus. L'arousal provoqu~ par les substances gustatives a ~t~ ~galement compar~ ~ celui dfi ~ l'eau. L'eau et les substances gustatives ont entrai'n~ des r~actions diff~rentes, eu ~gard aux param~tres consid~r~s. La plupart des substances gustatives o n t provoqu~ une r~ponse dose-d~pendante. La comparaison des r~sultats ECoG et comportementaux montre clairement que les substances gustatives entrafnant un comportement adversif ont provoqu~ un 'arousal' significativement distinct de celui produit par
J.E. STEINER, J. REUVENI
l'eau. Pour les r~actions de preference il n'a pas ~t~ observ~ de correlation avec les r~sultats ECoG. La possibilit~ d'utiliser la r~ponse d"arousal' comme indicateur objectif de l'aversion gustative est soulevde. The authors would like to thank Dr. Judith Ganchrow for critical reading of the manuscript.
References Carpenter, J.A. Species differences in taste preference. J. comp. physiol. Psychol., 1956, 4 9 : 1 3 9 144. Cohen, M.J., Landergen, S., Storm, L. and Zotterman, Y. Cortical reception of touch and taste in the cat. Acta physiol, scand., 1957, 40 (Suppl. 135): 1--50. Funakoshi, M. and Kawamura, Y. Summated cerebral evoked responses to taste stimuli in man. Electroenceph, clin. Neurophysiol,, 1971, 30: 2 0 5 - 2 0 9 . Funakoshi, M., Kasahara, Y., Yamamoto, T. and Kawamura, Y. Taste coding and central perception. In: D. Schneider (Ed.), Olfaction and Taste. Vol. IV, Wissenschaftliche Verlagsgesellschaft, Stuttgart, 1972: 336--342. Gentle, M.J. Taste preference in the chicken {Gallus domesticus L.). Brit. poult. Sei., 1972, 1 3 : 1 4 1 155. Grill, H. and Norgren, R. The taste reactivity test. I. Mimetic responses to gustatory stimuli in neurologically normal rats. Brain Res., 1978, 143: 2 6 3 279. Kim, J. and Kohout, F.I. Analysis of variance and covariance. In: H.N. Norma, H.C. Hull, K. Steinbrenner and D.H. Bent (Eds.), SPSS--Statistical Package for t h e Social Sciences, 2nd ed. McGrawHill, New York, 1976: 398--422. Longo, V. Electroeneephalographic Atlas for Pharmacological Research. Effect of Drugs on the Electrical Activity of the Rabbit Brain. Elsevier, Amsterdam, 1962. Monnier, M. and Gangloff, H. Atlas for Stereotaetic Brain Research on the Conscious Rabbit. Elsevier, Amsterdam, 1961, Pfaffmann, C., Erickson, R.P., Frommer, G.P. and Halpern, B. Gustatory discharges in the rat medulla and thalamus. In: W.A. Rosenblith (Ed.), Sensory Communication. M.I.T. Press, Cambridge, Mass., 1961: 455--473. Pfaffmann, C., Norgren, R. and Grill, H. Sensory affect and motivation. Ann. N.Y. Acad. Sci., 1977, 290: 18--34. Schwartzbaum, J.S. and Morse, J. Taste responsivity of amygdaloid units in behaving rabbit: a method-
DIFFERENTIAL AROUSAL
TO GUSTATORY
STIMULI
ological report. Brain Res. Bull., 1978, 3: 131-141. Steiner, J.E. The gustofacial response: observations on normal and anencephalic newborn infants. In: J.F. Bosma (Ed.), IV Symposium on Oral Sensation and Perception. U.S. DHEW, N.I.H., Bethesda, Md., 1973: 254--278. Steiner, J.E. Innate discriminative human facial expressions to taste and smell stimulation (a discussion paper). Ann. N.Y. Acad. Sci., 1974, 273: 229--233. Steiner, J.E. Facial expressions of the neonate infant indicating the hedonics of food-related chemical stimuli. In: J.M. Weiffenbach (Ed.), Taste and
11
Development. U.S. DHEW, N.I.H., Bethesda, Md., 1977: 173--188. Sudakow, K., MacLean, P.D., Reeves, A. and Marino, R. Unit study of exteroceptive inputs to claustrocortex in awake sitting squirrel monkey. Brain Res., 1971, 28: 19--34. Sulg, I.G. Mammal EEG analysis. Acta neurol, scand., 1989, 45: 431--458. Yamamoto, T. and Kawamura, Y. Summated cerebral responses to taste stimuli in rat. Physiol. Behav., 1972, 9 : 789--793. Yamamoto, T. and Kawamura, Y. Cortical responses to electrical and gustatory stimuli in the rabbit. Brain Res., 1975, 94: 447--463.
1
© Elsevier/North-Holland Scientific Publishers, Ltd.
DIFFERENTIAL AROUSAL RESPONSE TO GUSTATORY STIMULI IN THE AWAKE RABBIT J.E. STEINER and J. REUVENI
Department o f Oral Biology, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem (Israel) (Accepted for publication: October 19, 1978)
The perception of gustatory stimuli may be measured behaviorally or electrophysiologically. Although the sensory modality of taste may be subdivided into a relatively small number of quality categories, another split is apparent for these sub-modalities, namely the hedonic dimension, reflected behaviorally by indifference, acceptance or rejection (Pfaffmann et al. 1977). As contrasted with other sensory modalities only little is known about cortical taste mechanisms and their electrophysiological correlates. Information is mainly related to attempts to record single cell activity or evoked responses {Cohen et al. 1971; Funakoshi and Kawamura 1971; Sudakov et al. 1971; Funakoshi et al. 1972; Yamamoto and Kawamura 1972, 1975). A definitive statement of EEG correlates of gustation remains to be made. In the present experiments an ECoG study was undertaken to compare the arousal response induced by the intraoral presentation of deionized water, with those induced by identical presentation of several concentrations each of sweet, salty, sour and bitter stimuli. The unanesthetized and restrained rabbit was chosen for these experiments because: (a) the electrical brain activity of this animal is well known in different physiological and pharmacological conditions (Monnier and Gangloff 1961; Longo 1962); (b) the behavioral response of this species to tastants has been studied {Carpenter 1956; Ganchrow in preparation).
Methods
Records were obtained from 17 'chronic' preparations of male rabbits (Lepus europaeus) (2.6 kg average body weight}. Epidural silver screw recording electrodes were implanted according to the widely accepted stereotaxic method of Monnier and Gangloff {1961), under pentobarbital anesthesia (50 mg/kg, b.w.) and local anesthesia of the head and eye region (Lignocalne 1% solution infiltration). In all experiments the right eye was enucleated to permit free access to the region of the rhinal sulcus. Electrodes were implanted in the orbital wall and in the region of the limbic (A, B) and occipital cortex (C) as shown in Fig. 1 according to stereotaxic coordinates. In 7 of the animals the brain was later removed and the location of the electrodes verified. After surgery animals received appropriate local and systemic antibiotic treatment. Rapid recovery was confirmed by normal motor behavior as well as regular food and water intake. Taste experiments were carried out about 3--6 days after surgery in a shielded and air-conditioned laboratory. For the actual experiment the animal was placed in a special sling (Monnier and Gangioff 1961) which minimized body movements. No pharmacological agents were given. The sapid substances and their concentrations are presented in Table I. All chemicals were reagent grade and were dissolved in deionized water. For sour stimuli two different chemicals were chosen, acetic acid and ascorbic acid. The
2
J.E. S T E I N E R , J. R E U V E N I B
C
D
E
F
G
0 3 6
A
12
15 m Ttmm
SUTURA SAGITTALIS c
b
•
t
•
b
¢
0 [
>'---- c
I-i2":- 1 I ] Fig. 1. Location of the electrodes according to the stereotaxic coordinates of Monnier and Gangloff (1961). See text.
latter was selected in order t o sample a sour stimulus n o t causing simultaneous olfactory or trigeminal stimulation, as acetic acid m a y do. Taste stimuli as well as deionized water were delivered to the dorsal surface o f the tongue with the m o u t h closed, f r om outside the Faraday cage via a gravitational flow system for a fixed duration o f 5 sec and a fixed 10 ml volume. Water rinses lasted 20-25 sec and repose between stimuli at least 10 min. All stimuli were at r o o m t e m p e r a t u r e (23°C) and presented in a r andom i z e d sequence. In o r d er to diminish reflex swallowing during stimulus flow, the h a m m o c k was tilted slightly d o w n w a r d at t he head end, facilitating drooling o f the fluids o u t of the
m out h. Records were perform ed with a 12channel apparatus. The brain activity was simultaneously recorded on a 4-channel magnetic tape recorder. The arousal response appearing after water and sapid stimuli were evaluated according to the following 3 parameters: (a) Duration of arousal response was measured in seconds, from the onset o f the stimulus for as long as arousal was detectable by visual inspection o f the tracing. (b ) Change in frequency was determined by a modified technique based on Sulg (1969) for manual analysis o f frequencies. The resting trace was sampled during a 6 sec period prior to stimulus presentation. The frequency in this sample was c o m p a r e d with t hat for the 6 sec period starting from the 'stimulus off' mark of the actual presentation. The prestimulus frequency was arbitrarily labeled as 100% and the change was expressed as percentage increase. (c) Changes in amplitude were quantified from the brain activity recorded on magnetic tape. Tapes were analyzed by a POP-15/140 computer. The absolute average amplitude was determined for a 6 s e c prestimulus period. This value was again labeled arbitrarily as 100%. The absolute average amplitude o f a 6 sec poststimulus period was expressed as a percentage of the prestimulus amplitude. From the individual measures calculated in each o f the stimulus trials for each experimental animal and across stimulus types (tastes) mean values were calculated. The means obtained in those trials across all animals, where water was presented as stimulus (n = 60), served as control value. The duration of the arousal, the change in frequency and the change in amplitude induced by the tastants were com pared to the values obtained after water (control) stimulation. Furthermore, the means, across all trials used in each o f the p a r t i c u l a r stimulus concentrations, were also com pared in order to assess a doseresponse relationship. A c o m p u t e r i z e d program (ANOVA) for a two-way analysis of variance was used to
D I F F E R E N T I A L A R O U S A L TO G U S T A T O R Y STIMULI
assess the significance of the differences found between control and tastant stimulations as well as between the values found with different stimulus concentrations (Kim and K o h o u t 1976).
3
Deionized water was presented to all animals, with a total of 60 trials. This stimulus was found to induce an arousal reaction which lasted 14.0 (+ 2.3) sec; it caused a 4.7% increase of the ECoG frequency and a 25% amplitude reduction as compared to the prestimulus conditions. The values thus obtained are presented as control values in Tables II-VI.
across the different concentrations (Table III). It can be seen that t w o of the salt concentrations, that o f 1.0 and 2.0 M, induced an arousal significantly different from that induced by water, both in terms of duration and of amplitude depression. Comparing the arousal reactions induced by different NaC1 concentrations, it was found that increasing the concentration significantly prolonged the duration of the arousal. Comparing the frequency increment caused by increased NaC1 concentrations, one can see that the lowest salt concentration used, 0.1 M, produced an increment similar to that of water. But 0.5 M, 1.0 M and 2.0 M salt solutions induced markedly higher frequency increases than water or the weakest NaC1 solution did.
(b) Sweet
(d) Sour
Sucrose was used in concentrations listed in Table I in 14 experiments with a total of 57 trials across the different concentrations (Table II). It can be seen that the arousal reaction in response to different sucrose concentrations showed only a slightly significant difference from the reaction caused by deionized water ('tasteless' or control) stimulation.
Results were obtained for ascorbic acid in 11 experiments with a total o f 40 trials (Table IV). From this table it may be seen that stimulation with ascorbic acid induced a markedly different arousal from that caused by water. All b u t the weakest (0.01 M) concentration induced a significantly longer lasting arousal than did water. Frequency increments were observed for all but the weakest concentrations. The differences between the waterinduced frequency increment and that caused by the t w o highest ascorbic acid concentrations (0.3; 0.5 M) were found to be highly sig-
Results
(a ) Tasteless
(c) Salty NaC1 was used in concentrations listed in Table I to present salty stimuli to the animals, in 19 experiments with a total of 64 trials
TABLE I Stimulants and their concentrations (in M) presented intraorally. Modality :
Sweet
Salty
Sour
Stimulant:
Sucrose
NaCI
Aseorbic acid
Acetic acid
Quinine HC!
0.1 0.5 1.0 2.0
0.1 0.5 1.0 2.0
0.01 0.1 0.3 0.5
0.025 0.05 0.125
0.001 0.01 0.03 0.05
Bitter
4
J.E. S T E I N E R , J. R E U V E N I
T A B L E II Arousal r e s p o n s e to sucrose s t i m u l a t i o n . Parameters
R e s p o n s e d u r a t i o n in sec Mean S.D. F r e q u e n c y i n c r e m e n t in % (prestim. freq. = 100%) A m p l i t u d e d e c r e m e n t in % (prestim. amplit. = 1 0 0 % )
H20
14.0 (2.3) +4.7 --25
S t i m u l a n t c o n c e n t r a t i o n in M
Significance o f F in A N O V A test *
0.1
0.5
1.0
2.0
16.6 (3.2)
20.8 (4.4)
18.0 (3.3)
21.3 (2.3)
+10.2 --11
+4.6 --17
+13.9
+18.0
--28
--26
0.1--2
0.02
0.5--2
0.04
* Signs a n d n u m b e r s o n t h e left side o f this c o l u m n s h o w t h e pairs o f s t i m u l i c o m p a r e d . R i g h t side n u m b e r refers to level o f significance.
nificant. Furthermore, a gradual doseresponse relationship was apparent. The depression of amplitude also showed a similar trend of gradual effect. In 5 of the relevant comparisons, differences were found to be significant. Acetic acid was used in 9 experiments with a total of 37 trials {Table V). As Table V makes clear, acetic acid also induced a markedly different arousal from that caused
by water stimulation. The increasing concentrations of the stimulant resulted in a gradually prolonged duration of the arousal, as well as in an increment of frequencies. A similar trend could be observed for amplitude depression. The 0.125 M concentration of acetic acid was found to reduce the amplitude of the brain activity more markedly than did any other stimulant used in this experiment.
T A B L E III Arousal r e s p o n s e to NaCI s t i m u l a t i o n . Parameters
R e s p o n s e d u r a t i o n in sec Mean S.D.
F r e q u e n c y i n c r e m e n t in % (prestim. freq. = 100%) A m p l i t u d e d e c r e m e n t in % (prestim. amplit. = 100%)
H20
S t i m u l a n t c o n c e n t r a t i o n in M 0.1
0.5
1.0
2.0
14.0 (2.3)
13.0 (4.2)
19.1 (4.4)
19.7 (4.0)
34.8 (5.7)
+4.7
+2.2
--25
--30
+17.4
--28
+7.3
--34
Significance o f F in A N O V A test * H20--2 H20--1 0.1--2 0.5--2 0.1--0.5 l --2
0.002 0.001 0.001 0.03 0.02 0.007
+22.6
0.1--2
0.03
--43
H20--2 H20--1
0.05 0.05
* Signs a n d n u m b e r s o n t h e left side o f this c o l u m n s h o w t h e pairs o f stimuli c o m p a r e d . T h e r i g h t side n u m b e r refers t o t h e level o f significance.
D I F F E R E N T I A L A R O U S A L TO G U S T A T O R Y STIMULI TABLE IV Arousal response to ascorbic acid stimulation. Parameters
Response duration in sec Mean S.D.
Frequency increment in % (prestim. freq. = 100%) Amplitude decrement in % (prestim. amplit. = 100%)
H20
Stimulant concentration in M 0.01
0.1
14.0 (2.3)
11.44 (3.22)
19.28 (4.29)
37.22 (12.98)
61.23 (15.8)
+4.7
+3.0
+9.1
+19.7
--43
--23
--24
--23
0.3
0.5
Significance of F in ANOVA test * H20-0.5 H20--0.3 H20--0.1 0.01-0.5 0.01--0.3 0.1 - 0 . 5 0.1 - 0 . 3
0.001 0.001 0.03 0.001 0.001 0.002 0.03
+29.5
H20-0.5 H20-0.3 0.01-0.5
0.01 0.01 0.01
--47
H20-0.5 0.01-0.5 0.01-0.3 0.1 - 0 . 5 0.1 - 0 . 3
0.01 0.005 0.01 0.006 0.03
* Signs and numbers on the left side of this column show the pairs o f stimuli compared. The right side number refers to the level of significance.
(e) Bitter Quinine hydrochloride was used in concentrations shown in Table I to present animals with bitter stimuli, in 13 experiments with a
total of 47 trials. From the data presented in Table VI one can see that the bitter stimulant induced a clearly different arousal reaction from t h a t to water. For all 3 measured param-
TABLE V Arousal response to acetic acid stimulation.
Parameters
Response duration in sec Mean S.D.
Frequency increment in % (prestim. freq. = 100%) Amplitude decrement in % (prestim. amplit. = 100%)
H20
14.0 (2.3)
+4.7
--25
Stimulant concentration in M 0.025 (1%)
0.05 (2%)
0.125 (5%)
25.0 (7.4)
28.4 (6.8)
83.1 (17.4)
Significance o f F in ANOVA test *
H20--0.125 H20--0.05 H20--0.025 0.025--0.125 0.05 --0.125
0.001 0.001 0.001 0.003 0.001
+18.6
+24.8
+27.0
H20--0.125 H20--0.05
0.01 0.01
--37
--46
--51
H20--0.125 H20--0.05
0.001 0.006
* Signs and numbers on the left side of this column show the pairs of stimuli compared. The right side number refers to the level o f significance.
6
J.E. STEINER, J. R E U V E N I
TABLE VI Arousal response to quinine stimulation. H20
Parameters
Response duration in sec Mean
S.D.
F r e q u e n c y increment in %
Stimulant concentration in M 0.001
0.01
0.03
0.05
14.0 (2.3)
20.3 (5.4)
22.7 (7.8)
27.6 (6.3)
39.8 (11.8)
+4.7
+5.9
--25
--16
H20--0.05 H20--0.03 0.001--0.05 0.01 --0.05 0.001--0.03
0.001 0.001 0.006 0.02 0.05
+17.5
+28.8
+40.0
H20--0.05 H20--0.03 0.001--0.05 0.01 --0.05 0.03 --0.05
0.01 0.03 0.001 0.002 0.03
--25
--33
--44
H20-0.05 H20--0.03 0.001--0.05 0.001--0.03 0.01 --0.05
0.007 0.04 0.002 0.02 0.01
(prestim. freq. = 100%)
Amplitude decrement in % (prestim. amplit. = 100%)
Significance of F in ANOVA test *
* Signs and numbers o n the left side o f this c o l u m n s h o w the pairs of stimuli compared. The right side number refers to the level of significance.
eters there was a clear gradual effect as the concentration of the stimulant increased. Data related to stimulation with 0.03 and 0.05 M concentration o f quinine differ significantly from those related to the water stimulation. Furthermore, most comparisons
between the results obtained by increasing stimulus concentrations also reveal significant differences. To gain an overview, the measures of the responses to the lowest and highest concentrations of each stimulant are compared with
E.E.G. AROUSAL UESPORSEIRTHEAWAKE RAB811 TO THE LOWEST AND HIGHEST CONCERTRATIOHSOF TASTANTS Tastants: H20 SUCROSE
001
--1
concentration
Highest
concentration
~
o i L\\\~\,~.~.z~x~\~--
:=
zo
28
Lowest
r--
G001 O U I N I N E ' H C [ DOS
t i l
ASCORBIC ACID o~o
ACET,C AC,D ~
l I
0
i r,
L ~ ,
,
I,
, ,
0.5
AMPLITIJBE RATIO POST-STIMI PRE-STIM
;
P I0
0
50 100 0 1 30 0 50 RESPOIfSE TIME (see) FREO,UEN£Y INCREASE{'/.) [RELATE{] TOSPONTANEOUSA['TIWTY}
Fig. 2. Comparative bar-graph of the values obtained in all 3 parameters used for the quantification of the arousal response. Concentrations ot tastants in M .
DIFFERENTIAL AROUSAL TO GUSTATORY STIMULI measures of water response in Fig. 2. It shows that: (a) Duration o f the arousal reaction induced by the lowest concentration of all sapid substances given in this experiment did n o t differ significantly from that induced by water (tasteless). The highest concentrations of the tastants, however, elicited a markedly longer lasting arousal than did water. Except for sucrose, this difference was found to be significant, the longest lasting arousal was observed in response to stimulation with 0 . 1 2 5 M acetic acid. (b) The increment in frequency caused b y the lowest concentrations of the tastants applied did n o t reveal any difference from that induced by water, with the exception of acetic acid in 0.025 M concentration, b u t even this was n o t significant. By contrast, the highest stimulus concentrations all caused a marked frequency increase as compared to water. Responses to bitter and sour stimuli differed significantly, while sweet and salty responses were only tending towards significance. 0.05 M quinine hydrochloride induced the highest frequency increment. (c) The decrement in amplitude caused by the lowest concentrations of the stimulating c o m p o u n d s was similar to that induced b y water. Only the lowest acetic acid concentration induced a slightly more detectable b u t still not significant amplitude decrem e n t as compared to the water effect. The highest concentrations of the salty, sour and bitter stimulants induced more marked amplitude decrements than did water or sucrose. 0.125 M acetic acid was found to have the most effect.
Discussion Findings presented here demonstrate that intraoral taste stimulation o f the adult restrained but non-anesthetized rabbit induced EEG arousal. The observed arousal response was f o u n d to be different when water was used as stimulant from that which appeared following taste stimulation. In response to sour and bitter tasting substances
7 the arousal showed a dose-dependent gradual increase, as indicated by 3 different quantifying parameters. These dose-dependent effects were expressed to a lesser degree with salty stimuli and were found n o t to be significant with sucrose. Ranking the effectiveness of sapid stimuli in inducing ECoG arousal, differentiable from that induced by water, gave the following order: quinine > acetic acid > ascorbic acid > NaC1 > sucrose. The results of the present study may be interpreted as reflecting in a quantitative manner (encephalographieally) the hedonic aspect of the tasting experience. In psychophysical testing situation, hedonics may be rated using the t w o bottle preference test. In this procedure the relative liquid consumption is used to assess taste preference (acceptance and rejection). In this testing situation it can easily be determined whether a certain tastant is perceived different from water in a positive or negative way. Comparing the data from behavioral testing (two bottle preference test} with the ECoG parameters reported here, one can conclude that tastants and their concentrations which were found aversive in the behavioral test, produced a more marked ECoG arousal than those which were preferred. Rabbits clearly discriminate between water and sucrose at 0.01 M concentration. A definite preference toward this tastant was observed at 0.1 M concentration. At the concentration of 1.0 M the animals again equally consumed sucrose and water while concentrations higher than 1.0 M were clearly rejected (Carpenter 1956). From the results of the present study it can be seen that 0.5 M sucrose markedly enhanced the duration o f the arousal as compared to the water response. Those concentrations, which were found in the mentioned behavioral study to be rejected, led to a certain increase in frequency. Although these differences were n o t found to be significant, y e t the ECoGs indicate an increase, although not a dramatic one, in vigilance and awareness, presumably reflecting aversion or dislike. It
8
seems that the dislike of the animals for the above-optimal concentrations of sucrose is more clearly measurable in the behavioral response than from the parameters of EEG arousal. Salty, sour and bitter stimulants in certain concentrations were found to be rejected by the rabbit in the psychophysical testing situation (Carpenter 1956; Ganchrow, submitted for publication). The ECoG data obtained in this study reveal marked alertness and arousal towards that particular range of concentrations which was found to elicit behavioral rejection in this species. From behavioral data it is known that rabbits discriminate between water and 0.01 M NaC1. Preference was observed around 0.1 M. A clear rejection was observed at 0.5 M. The behaviorally preferred salt concentrations caused an arousal reaction which was n o t significantly different from the water response, while the arousal induced by NaC1 in concentrations above 0.5 M produced a significantly different arousal from that induced b y water. The unpleasantness of the 0.5, 1.0, and 2.0 M salt solutions is reflected b y the duration of the response, as well as by frequency increment and amplitude decrement. One may conclude that the unpleasantness of the salty taste causes a marked b u t still not drastic arousal effect. Behavioral data indicated that rabbits did not differentiate ascorbic acid from water at concentrations o f 0.001 M and 0.01 M, while they clearly rejected this c o m p o u n d in 0.02 and 0.05 M concentrations (Ganchrow, manuscript submitted). Comparing the behavioral and arousal data, it can be stated that the behaviorally rejected concentrations of ascorbic acid induced indeed an arousal which differed significantly from the water response. Thus once again intensity of the arousal may be interpreted as an objective sign of tasteaversion. Acetic acid was found n o t to be discriminated from water b y rabbits in concentration of 0.0001 M. A clear behavioral rejection was observed in 0.001 and in 0.01 M concentra-
J.E. STEINER, J. REUVENI
tion (Ganchrow, manuscript submitted). The arousal data evidenced very markedly and significantly different responses from the waterinduced arousal, with 0.025 M up to 0.125 M concentration of this c o m p o u n d . The intensity of the arousal response was found dosedependent. One can, therefore, consider these parameters as an objective indication of taste aversion. Quinine was found in the present experiment to be the most effective sapid stimulant producing an arousal response. Behavioral data (Carpenter 1956) showed clear rejection of this c o m p o u n d in concentrations above 0.0001 M. The dose-dependent EEG findings obtained in the present study strongly support the assumption that the marked arousal, highly significantly different from the water response toward increasing concentrations of this substance, is indicative of taste aversion. Searching the relevant literature, no experimental study showing similar results could be found regarding any mammalian species. Yet, in one avian species, namely the chick (Gallus domesticus L.) an effort was made to use the length (duration) of EEG arousal to indicate objectively behavioral preference and rejection (Gentle 1972). It was found that many of the solutions which did n o t induce any behavioral response in the birds gave no EEG indications. Some acids were found to produce EEG reactions differentiable from those caused by water, even without concomitant behavioral responses. It was concluded that the duration of the EEG response in the chick may well be considered as a more sensitive detection technique of taste rejection than behavioral testing. As to the other animal experiments attempting to define a correlation between behavioral and electrophysiological data, it is perhaps worthwhile to note that a comparison of the firing rates of primary and secondary taste neurons with behavioral acceptance and aversion of salty taste in the rat, did not reveal any typical change in firing rate which might correspond to behavioral aversion (Pfaffmann et al. 1961).
D I F F E R E N T I A L A R O U S A L TO G U S T A T O R Y STIMULI
Most recently a study was reported on taste-induced activity of cells in the amygdala of the awake 'behaving' rabbit (Schwartzbaum and Morse 1978). In this experiment cells were found in n. centralis region of the amygdala to receive taste information. Analysis of the response pattern revealed evidence of units responding exclusively or highly differentially to tastants, presented as intraoral stimuli. These findings strongly suggest that it is possible to find, at the cellular level, physiological correlates indicating qualitative and motivational (hedonic) differences between different sapid stimuli. To date no study has been reported correlating EEG parameters to hedonic ratings b y humans. However, a summated cortical evoked response in man, due to intraoral water and sapid stimulation, was described (Funakoshi and Kawamura 1971) as being of two components. The first reflected water and apparently originated from tactile stimulation of the oral cavity; the second, more delayed, c o m p o n e n t o f the response was interpreted by these authors as strongly suggestive of reflecting the taste aspect of the intraoral stimulus. This part of the evoked response was also f o u n d to be concentrationdependent. In conclusion: it is perhaps n o t unjustified to assume that careful analysis o f tasteinduced EEG changes m a y become an objective indicator for both the relative intensity and hedonic dimension o f a perceived taste sensation. The findings of the present study strongly suggest such a possibility. One can also assume that the aversiveness of the unpleasant stimulus causes a more easily detectable reaction than pleasant stimuli may cause. In this c o n t e x t it is appropriate to mention that oro-facial m o t o r reactions of innate and reflex-like character were demonstrated in response to tastants as well as to some food-related odorants in the neonate human infant (Steiner 1973, 1974, 1977). Similar reactions were later also f o u n d in animals (Grill 1978; Ganchrow et al. manuscript submitted). These observations showed
9
that sweet stimuli and pleasant food odors induced more delicate and moderate responses, whereas sour and bitter tastants and offensive food odors always elicited quick dramatic and massive muscle reactions. It was concluded from these studies that pleasant and agreeable chemical stimuli do n o t carry any message o f urgency, while to the unpleasantness of sour and bitter stimuli and to the offensive f o o d odor the aspect of alarming urgency is attached. The negative hedonic aspect of such taste and odor stimuli is then evaluated by the central nervous system as an alarming sign, activating neural mechanisms of avoidance, defense and withdrawal. One may, therefore, state that just as offensive taste and smell stimuli activate more dramatic m o t o r reflexes than pleasant ones, so aversive tastants induce more drastic EEG signs of awareness and vigilance than indifferent or pleasant sapid materials do. In order to reach conclusive results, further experimentation is necessary using both different species of animals and human examinees. In such experimental conditions a correlation might be established between objectively detectable EEG signs and psychophysical rating and ranking of relative intensity and hedonics of perceived taste sensations.
Summary ECoG arousal response as elicited by deionized water and by several concentrations of sweet, salty, sour and bitter tasting substances, all applied intraorally, were studied in the awake restrained rabbit. The study was carried o u t on 17 chronic preparations o f adult male animals (2.6 kg average b o d y weight). Water as a stimulant was presented in 60 trials and tastants across qualities and concentrations in 245 trials. Arousal was quantitatively characterized b y its duration, frequency increment and amplitude decrement as compared to prestimulus conditions. Arousal induced b y tastants was
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compared to that induced by water. Water and tastants induced arousals differentiable by the parameters measured. Further, most tastants produced a dose-dependent response. Comparison of ECoG and behavioral data clearly indicate that tastants inducing behavioral aversion also produce an arousal which significantly differs from response to water while behavioral preference was found not to show similar correspondence with ECoG data. The possibility of using arousal as an objective indicator for taste aversion is discussed.
R~sum~ Activations ECoG spdcifiques aux stimulus gustatifs chez le Lapin dveilld On a ~tudi~, chez le Lapin ~veill~ mais en contention, les activations ECoG suscit~es soit par de l'eau d~sionis~e, soit par des solutions en diverses concentrations, de substances sucr~es, salves, acides et am~res, toutes appliqu~es dans la cavit~ buccale. L'analyse a port~ sur 17 animaux, m~les adultes, en preparation 'chronique' (poids m o y e n 2,6 kg). L'eau a ~t~ utilis~e en tant que stimulant au cours de 60 essais et les diff~rentes substances gustatives dans 245 essais. La r~action d"arousal' a ~t~ quantitativement appreci~e par la dur~e, par l'augmentation en fr~quence et la diminution d'amplitude qui l'accompagnait, et compar~e l'activit~ pr~stimulus. L'arousal provoqu~ par les substances gustatives a ~t~ ~galement compar~ ~ celui dfi ~ l'eau. L'eau et les substances gustatives ont entrai'n~ des r~actions diff~rentes, eu ~gard aux param~tres consid~r~s. La plupart des substances gustatives o n t provoqu~ une r~ponse dose-d~pendante. La comparaison des r~sultats ECoG et comportementaux montre clairement que les substances gustatives entrafnant un comportement adversif ont provoqu~ un 'arousal' significativement distinct de celui produit par
J.E. STEINER, J. REUVENI
l'eau. Pour les r~actions de preference il n'a pas ~t~ observ~ de correlation avec les r~sultats ECoG. La possibilit~ d'utiliser la r~ponse d"arousal' comme indicateur objectif de l'aversion gustative est soulevde. The authors would like to thank Dr. Judith Ganchrow for critical reading of the manuscript.
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