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The role of olfactory and orosensory factors in the alcohol preference of inbred strains of mice
Physiology and Behavior, Vol. 6, pp. 53-95. Pergamon Press, 1971. Printed in Great Britain
The Role of Olfactory and Orosensory Factors in the Alcohol Preference of Inbred Strains of Mice' MARVIN
NACHMAN
Department of Psychology, University of California, Riverside, California, U.S.A. AND CHR1STIANE LARUE AND JACQUES LE MAGNEN
Laboratoire de Neurophysiologie, Colldge de France, Paris, France (Received 7 July 1970)
NACHMAN,M., C. LARUE AND J. LE MAGNEN. The role of olfactory and orosensory factors in the alcohol preference
of inbred strains of mice. PHYSIOL. BEHAV. 6 (1) 53--59, 1971.--Removal of the olfactory bulbs eliminated the aversion to alcohol which is seen in normal BALB/c mice but did not abolish the preference for alcohol seen in normal C57BL mice. This result, along with the fact that BALB/c mice appear to avoid alcohol immediately, without prior experience, led to the hypothesis that BALB/c mice are more responsive than are C57BL mice to alcohol as a sensory stimulus. To test this hypothesis, a conditioning procedure was used in which normal animals of both strains drank alcohol in a single bottle test and were then injected with toxic lithium chloride. It was found that BALB/c mice learned aversions to alcohol, saccharin, and sucrose, whereas C57BL mice learned aversions to saccharin and sucrose but were deficient in learning an alcohol aversion. It was concluded that BALB/c mice normally avoid alcohol because of its odor but that for C57BL mice, other sensory cues or post-ingestional factors play an important role in their preference for alcohol. Olfaction Orosensory factors Alcohol preference Mouse strains Olfactory bulbs factors Taste preference Learned aversions Conditioned taste aversions
Post-ingestional
significant role in strain differences in alcohol selection. Perhaps most important is the fact that the strain differences can be seen on the first day of testing with the solutions, a finding which suggests that these differences may exist before learning due to post-ingestive factors has occurred. The role of taste in alcohol intake is further indicated by the fact that alcohol intake in mice can be increased by sweetening the solutions with sucrose [16] and that in rats, a positive correlation exists between quinine aversion thresholds and alcohol intake [8]. The specific influence of olfaction in alcohol selection has also been demonstrated by the finding that bulbectomized rats, in comparison with normal rats, show less of an alcohol preference at low alcohol concentrations and less of an alcohol aversion at high concentrations [5]. Elimination of oropharyngeal sensations by directly infusing the alcohol into the stomach has also been shown to result in increased alcohol intake [1]. The present experiments used the high alcohol preferring strain, CB57BL, and the low alcohol preferring strain, BALB/c, and the first experiment was designed to determine
INBRED strains of mice differ greatly in the degree to which they will ingest alcohol. When given an ad lib choice between 10% v/v alcohol and water, for example, C57BL mice will show a strong preference for the alcohol solution whereas the BALB/c or DBA/2 strains will ingest very little alcohol [9, 101. While it is evident that these strain differences in alcohol intake are genetically determined, it is not known what physiological differences in the strains are responsible for the differences in alcohol selection. Self-selection of foods and fluids is often determined both by sensory factors and by postabsorptive effects. In the case of alcohol selection, there has been considerable research attempting to relate the strain differences in alcohol intake to strain differences in alcohol metabolism or toxicity [6, 17, 19]. However, there has been very little work reported which has attempted to relate strain differences in alcohol intake to possible gustatory or olfactory factors. Several lines of evidence, although each inconclusive, are suggestive of the hypothesis that sensory factors may play a
1This research was supported in part by NIH Special Research Fellowship (to M.N.) 7 F03 MH14939-01A1 and was conducted at the Coll6ge de France. 53
54
NACttMAN, LARUE ANt) IL MAGNI:N
whether removal of the olfactory bulbs would effect the strain differences in alcohol intake which normally exists in these mice. EXPERIMENT
1
Method The mouse strains were obtained from the animal laboratory of Orl~ns-La Source, France. The animals were 30 male C57BL/6 and 30 male BA_LB/c mice, which ranged in age from 8 to 13 weeks when testing began. The olfactory bulbs were bilaterally removed, under nembutal anesthesia, for 15 mice of each strain, while the other 15 mice of each strain served as unoperated controls. The bulbectomies were performed with suction and care was taken to entirely remove the olfactory bulbs. After a one week operative recovery period, all mice were given an ad lib two-bottle choice between a 10% v/v ethyl alcohol solution and tap water. The mice were caged individually in cages 16 × 16 × 20 cm and food pellets were always available. The test bottles used were 25 ml graduated cylinders with stainless steel spouts protruding 4 cm into the cage and separated by about 4 cm. The twobottle choice test was carried out for 12 days with the amount ingested measured daily; no attempt was made to correct for evaporation or drippage which was estimated to be relatively little, on the order of 0.1-0.2 ml per day. The bottles were refilled and their positions switched every 3 days. Alcohol preference scores were obtained daily and expressed as a percentage by dividing the alcohol intake by the total fluid intake and multiplying by 100. For each mouse, the alcohol containing bottle and stainless steel spout and the water bottle and stainless steel spout were kept constant throughout the 12 days. After this test period, stainless steel spouts were switched on several occasions to determine whether a particular spout rather than the test solution might be determining the preference. In the case of one mouse, a BALB/c bulbectomized, it was evident that the animal's large alcohol intake was determined by differences in the flow of the two spouts and the data of this animal were discarded. In addition, one C57BL bulbectomized mouse died during the experiment leaving 14 bulbectomized and 15 controls of each strain. As will be seen in the results below, the data of the first 12 days of testing indicated that the BALB/c controls showed the typical alcohol aversion which other investigators have previously reported, but that the bulbectomized BALB/c mice did not show any apparent aversion to alcohol. These results were found using a standard procedure of changing the position of bottles every 3 days, however, and this may have made it difficult for the bulbectomized BALB/c mice to locate the position of the alcohol and water bottles. Also, these results were found in the first 12 days of testing of bulbectomized and normal mice which had not previously had experience with alcohol solutions. To examine whether these results were limited to these test conditions, additional alcohol vs. water testing was done with less changing of bottle positions and using bulbectomized BALB/c mice which had previously shown an aversion to alcohol when they had been tested as normals. Eight of the control BALB/c mice, randomly selected from those which had shown a clear aversion to alcohol in the tests of the first 12 days, were bilaterally bulbectomized after they had had 21 days of alcohol vs. water testing. They were maintained, without interruption, for an additional 12 days of alcohol vs. water choice in which the bottles were changed
every 3 days and then tested for 24 days during which the position of the bottles was changed only once, after 12 days. For comparison, 6 of the BALB/c mice which had been bulbectomized before any testing, were randomly selected, maintained for the same total of 33 days of alcohol vs. water choice with the bottle positions changed every 3 days, and then also tested for 24 days of alcohol vs. water choice with bottle positions changed once after 12 days. In addition, 14 C57BL mice were tested in exactly the same way as the 14 BALB/c mice, with 8 of the C57BL mice having been bulbectomized after first serving as controls and the remaining 6 C57BL mice serving as bulbectomized from the beginning of the experiment. Because of drainage of one bottle, data were recorded from 14 BALB/c and 13 C57BL mice during the 24-day test. At the end of testing, the mice were sacrificed and the brains examined for the extent of the olfactory bulb ablations. A sagittat brain section of a normal and of a bulbectomized BALB/c mouse is presented in Fig. 1. Results The mean of the daily alcohol preference scores for each mouse for the first 12 day test period is presented in Fig. 2. For the C57BL strain, removal of the olfactory bulbs had no apparent effect on the overall 12-day preference for alcohol; the mean alcohol preference score was 73.8 per cent for the C57BL controls and 76.2 per cent for the C57BL bulbectomized mice. In contrast, the mean alcohol preference score for the BALB/c controls was 17.9 per cent which was significantly less than the mean score of 47.5 per cent for the BALB/c bulbectomized group (p < 0.01, all ps reported in Experiment 1 are for Mann-Whitney U-tests). The difference in alcohol intake between BALB/c controls and BALB/c bulbectomized mice was clearly evident on the very first day of testing. On this day, the BALB/c controls showed a strong alcohol aversion with 14 of the 15 mice drinking less alcohol than water and the mean intake for all 15 was 0.66 ml of alcohol compared to 3.51 ml of water. A further way of indicating the very small amounts of alcohol taken by BALB/c controls on the first day is to examine the
3 Control
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FIG. 2. Alcohol preference scores of individual control and bulbectomized mice of each strain during the first 12 days of two-bottle alcohol vs. water preference tests.
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FIG. 1. Unstained sagittal sections of a normal mouse brain (A) and of a brain from which the olfactory bulbs had been removed (B).
(facing page
54)
OLFACTORY, OROSENSORY FACTORS AND ALCOHOL PREFERENCE scores of individual animals. Of the 15 BALB/c controls, 13 drank 0.09 ml or less of alcohol and of these, 7 drank 0.4 ml or less of alcohol. Considering that spillage and evaporation probably accounted for about 0.1 or 0.2 ml of these amounts, it can be seen that BALB/c control mice ingest very little alcohol when first exposed to it in a choice situation. In contrast, the BALB/c bulbectomized mice showed no evidence of an alcohol aversion on the first day, the mean intake of this group on the first day was 1.7 ml of alcohol and 1.3 ml of water and the alcohol preference scores of all mice were distributed throughout the range between 21 and 81 per cent alcohol preference. The first day intake scores of the C57BL mice also revealed a significant difference between the controls and the bulbectomized (p < 0.05). In this case, however, the controls appeared to be relatively indifferent to alcohol with a mean preference score of 50.9 per cent and only 8 of the 15 control mice drank more alcohol than water, while in comparison, the bulbectomized C57BL mice had a mean preference score of 72.0 per cent on the first day and 13 of the 14 mice drank more alcohol than water. The control C57BL mice progressively increased their alcohol preference during the first few days to equal that of the bulbectomized C57BL mice. F o r example, on the second, third and fourth days of testing, the number of control C57BL mice which drank more alcohol than water increased to 9, 12 and 14 animals, respectively. The data of McClearn and Rogers [9] also indicate that C57BL mice do not show an alcohol preference on the first day and Rodgers [14] states that initial establishment of stabilized preferences in mice requires 3 or 4 days. Figure 3 contains the alcohol preference scores of the bulbectomized mice of each strain which were tested for 24 days with the bottle positions changed only once. These results are essentially the same as those seen for the bulbectomized mice in Fig. 2, i.e. the bulbectomized C57BL mice show a clear alcohol preference, with a mean alcohol preference score of 81.9 per cent and the bulbectomized BALB/c mice drink approximately equal amounts of alcohol and water, with a mean alcohol preference score of 55.9 per cent. Thus, the fact that the animals were more experienced by the time of the 24-day tests or that bottle positions were changed less often had no apparent influence on the alcohol preference scores. Also, there was no evidence of any trend within each 12-day segment. Figure 3 contains both the data of animals which had been bulbectomized at the beginning of the experiment as well as those which had first served as controls
C 57 BL/6
32]
rl " ~b
io
AlCOhOl
30
~
preference
" fro " 6b
I percent
" 70
" 8o
of total
ab
t~0
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FIG. 3. Alcohol preference scores of individual bulbectomized mice of each strain during 24-day tests in which bottle positions were changed once after 12 days. The shaded portions represent animals which had been tested before and after they were bulbectomized and the unshaded portions represent animals tested only after bulbectomy.
55
and it was found that these subgroups did not differ in their alcohol preference scores (.as > 0.05). In the case of the BALB/c mice, this indicates that the lack of aversion to alcohol following the removal of the olfactory bulbs was not influenced by whether or not the mice had previously had experience with avoiding alcohol.
Discussion It is likely that the effects of the bulbectomy operations in this study were due specifically to the removal of the olfactory bulbs. Rodgers and McClearn in a review article [15] cite experiments in which removal of only the olfactory bulbs produced inconclusive effects on the alcohol aversion of mice of the A strain but that removal of the anterior third of the cerebrum including the olfactory bulbs produced increased alcohol intake in both A and BALB/c mice. In the present study, frontal damage was typically slight, as illustrated in Fig. 1. The lesions in general were restricted to the olfactory bulbs and there was no evidence of a relationship between alcohol preference scores and extent of lesion. The alcohol preference scores indicate that olfactory mechanisms play an important role in the normal avoidance of alcohol by the BALB/c strain. The bulbectomized BALB/c mice appear to be relatively indifferent to alcohol as judged by the fact that as a group they drank approximately equal amounts of water and alcohol and all animals ranged between alcohol preference scores of 26.4 and 77.9 per cent. However, although the animals did not show strong aversions, they apparently could discriminate between the alcohol and water bottles as revealed by the day to day intake scores which show that some animals have a small but consistent daily preference for either the alcohol solution or the water. Furthermore, the evidence indicates that the lack of aversion to alcohol by the bulbectomized BALB/c mice is not due to an inability to locate the position of the alcohol bottle since the BALB/c bulbectomized mice still did not show any sign of avoiding alcohol when bottle positions were kept constant for 12 days, even if the animals had previously had experience with avoiding alcohol when they were controls. On the basis of these results, it seems justifiable to conclude that the BALB/c mice normally avoid 10 per cent alcohol because of the odor of this substance and that in the absence of the sense of smell, the amount of alcohol ingested does not have aversion producing effects. This conclusion differs, of course, from those which relate an animal's limited alcohol intake to a metabolic tolerance factor [3] or to the capacity to metabolize alcohol as shown by acetaldehyde levels or by limiting amounts of liver alcohol dehydrogenase enzyme [14]. While it is possible that both olfactory and metabolic factors may be operating, the present results suggest that metabolic factors are not principally responsible for limiting the intake of alcohol in the BALB/c mice. If the mice were avoiding alcohol because of some toxic metabolic consequence, it would be difficult to understand why removal of the olfactory bulbs would permit the animals to ingest larger amounts of alcohol without producing an aversion due to the same toxic consequences. The small amounts of alcohol ingested by BALB/c mice on the first day of testing provides further support for the idea that sensory factors rather than metabolic factors are directly responsible for the observed alcohol aversion. Although it is certainly possible that a learned aversion could have occurred within the first test day, the amounts of alcohol ingested were extremely small, and were much less than estimates of the amounts that can be metabolized in 24 hr [16]. These results
56
N A C t t M A N . LAR[)[; A N I ) 11
suggest that the animals were responding directly to the smell or taste of the alcohol rather than avoiding it because of some consequence of intake. It should be noted, however, that the possible existence of strain differences in sensory response to alcohol in no way negates or argues against the concomitant existence of strain differences in metabolic capacity. In fact, it may be that sensory differences have evolved because of their adaptive value in relation to metabolic differences. The C57BL mice differ in several important ways from BALB/c mice in their intake of alcohol and it is likely that differing physiological substrates may be involved in influencing alcohol intake in the two strains, a suggestion which has arisen as a result of genetic analysis of the strains [4]. The most obvious difference between the strains is that BALB/c mice avoid alcohol, while the C57BL mice show a strong preference for alcohol over water. Second, the alcohol aversion shown by BALB/c mice depends on the olfactory bulbs whereas the preference for alcohol by C57BL mice is maintained after removal of the olfactory bulbs. Third, the aversion to alcohol shown by the BALB/c mice seems to be immediate and apparently does not have to be learned whereas the preference for alcohol by C57BL mice is not seen immediately and may take several days to develop. Fourth, it is likely that C57BL mice have a much higher threshold for preferring alcohol than BALB/c mice have for avoiding alcohol, since large differences in these thresholds have been reported between C57BL mice and another alcohol avoiding strain, DBA/2 [18]. The fact that BALB/c mice show an immediate aversion to alcohol and this aversion is eliminated by bulbectomy suggests that the animals are responding to the odor of alcohol as an aversive stimulus. In contrast, the high threshold at which C57BL mice first select alcohol, along with the fact that the alcohol preference remains after bulbectomy, suggests that C57BL mice are either relatively insensitive to the odor of alcohol or that if sensitive, the odor is much less noxious and can be readily overcome by some positively reinforcing aspects of the alcohol. An examination of the first day alcohol preference scores in normal and bulbectomized C57BL mice gives some support for the idea that C57BL mice may have a slight negative response to the odor of alcohol. Whereas the normal C57BL mice showed no preference for alcohol over water on the first day and gradually increased their preference over days, the bulbectomized C57BL mice preferred alcohol from the first day. Also, the first day data of McClearn and Rodgers [9] indicates a small aversion to alcohol by normal female C57BL mice which later becomes a preference.
~IA(iNI N
solution was used because it was believed that a It) per cent solution might be strong enough for both strains to learn the aversion and thus obscure the difference between the strains. Method The animals were 30 male C57BL and 30 male BALB/c mice ranging in age from 7 to 14 weeks and obtained from the same supplier as in Experi,nent 1. They were housed in individual cages with food pellets always available. For each strain, mice were assigned randomly to an experimental grot,p (n 20) or to a control group (n := I0). The testing procedure was modified slightly from one previously used with rats to produce learned alcohol aversions [11]. All animals were given a single water bottle in their home cage for 1 hr daily for 3 days and the amount ingested in the first 10 min was measured. On the 4th day, the treatment day, all animals were given a single-bottle 10-min test with 6.7 per cent v/v ethyl alcohol. Three rain after the alcohol test, the experimental groups of each strain were injected intraperitoneally with 0.02 ml per g body weight of 0.15 m LiCI, while the control animals were not treated. The time interval of 3 rain was chosen arbitrarily although it was known to be well within the effective times to produce learned aversions in rats. On the two successive days, Days 5 and 6, the animals were given access to water for one hour to extinguish any generalized aversion to drinking and on Day 7, the test day, they were again given a single-bottle 10-rain test wilh 6.7 per cent ethyl alcohol and their intake recorded. The results showed that the experimental groups of the two strains did differ in the degree to which they avoided alcohol on the test day. To determine whether this strain difference could be attributed to the alcohol stimulus or whether a similar strain difference would occur with other substances, the same experimental procedure was then repeated in a second part of the experiment using 0.I per cent saccharin solution as the conditioned stimulus instead of alcohol. The same animals were retested as experimental and control animals and the second part of the experiment was started between 10 and 30 days after the end of the first part. During this time interval the animals were kept on ad lib food and water. The sequence of testing in the second part of the experiment was identical to that of the first part: on the first three days, experimental Days 8-10, the mice received 1 hi" water daily; on Day 11, they received 0.t per cent sodium saccharin for 10 min and 3 rain later the experimental groups were injected with LiCt; on Days 12 and 13 the mice received 1 hr water daily and on Day 14 they were ~ested for I0 rain with 0.1 per cent saccharin.
EXPERIMENT 2
One of the conclusions of Experiment 1 was that C57BL mice may be less responsive than BALB/c mice to sensory stimulating properties of alcohol. Experiment 2 was designed to further test this conclusion by using a conditioning procedure in which the ingestion of alcohol was followed by a toxic injection of lithium chloride and the animals subsequently tested for their learned aversion to ingesting alcohol. It was hypothesized that BALB/c mice would readily learn to avoid ingesting alcohol but that C57BL mice would be relatively deficient in such learning. Similar conditioned aversions to ingesting alcohol have been reported in rats [11] and in mice [12], and it has also shown that the salience of an ingested solution is an important predictor of taste-aversion learning [7]. In Experiment 2, a 6.7 per cent v/v ethanol
Results and Discussion Figure 4A contains the amount of alcohol drunk on the test day, Day 7, for the experimental and control groups of each strain. It can be seen that the BALB/c mice had learned the alcohol aversion since the 0.48 ml drunk by the experimental group was significantly less than the 1.I 3 ml drunk by their controls (p < 0.001, all ps reported in Experiments 2 and 3 are for two-tailed t tests). In contrast, the 0,86 ml of alcohol drunk by the experimental C57BL mice, while slightly less, was not significantly different from tbel 0.98 ml alcohol drunk by their controls (p > 0.2). On Day 4, before the LiCI treatment, the BALB/c mice showed no evidence of an alcohol aversion. The alcohol intake of BALB/c mice was 1.24 ml which was not significantly
OLFACTORY, OROSENSORY FACTORS AND ALCOHOL PREFERENCE
ml 2,00. ~
control experimental
1.50 1,00 0,50.
BALB/c
C 57BL
ALCOHOL
BALB/c
C 57 BL
(~SACCHARIN
FIG. 4. Mean (~: S.E.) amount (ml) of alcohol (A) and of saccharin (B) drunk on the test day by control and experimental mice of each strain.
different (p > 0.2) than the 1.08 ml alcohol intake of C57BL mice and in both strains, the amount of alcohol ingested on Day 4 was almost identical with the amount of water ingested in 10 min on Day 3. This was probably due to the use of a single-bottle forced-choice alcohol test as well as to the weaker 6.7 per cent alcohol used. F o r both strains, there were no significant differences in alcohol intake of control animals between Day 4 and Day 7 (ps > 0.2). The alcohol conditioning procedure did not have any apparent effect on the saccharin intake scores of Day 11 ; on this day, there were no significant differences between experimental and control mice of either strain in saccharin intake (ps > 0.1). Figure 4B contains the saccharin intake scores for both strains on the test day, D a y 14. In contrast to the results with alcohol, it can be seen that the C57BL mice learned the saccharin aversion as well as, if not somewhat better than did the BALB/c mice. The experimental BALB/c mice drank 1.29 ml of saccharin on Day 14 which was significantly less than the 1.72 ml drunk by their controls (p < 0.05) and the experimental C57BL mice drank 0.87 ml which was much less than the 1.76 ml drunk by their controls (p < 0.001). On Days 5 and 12, the days immediately following each of the LiCI treatments, the 10-min water intake of the experimental animals of each strain was about 15 per cent less than the controls. This decrease in water intake reflected either a generalized aversion to drinking or a residual effect of the LiCI sickness. However, by the next day in each case, Day 6 and Day 13, there was no longer a difference between the water intake of the experimentals and controls of either strain in the first 10 min or in the 60 min totals. Thus, the aversions shown to alcohol on D a y 7 and to saccharin on D a y 14 could not be attributed to any residual sickness from the LiCI but were specific to the ingested solutions. The alcohol conditioning results of Experiment 2 showed that C57BL mice did not learn an alcohol aversion as well as BALB/c mice. The saccharin tests, by finding that both groups did learn a saccharin aversion, demonstrated that the strain differences in learned alcohol aversion was not due to some extraneous variable such as learning ability or sensitivity to LiCI but was specific to alcohol. However, one aspect of the Experiment 2 results requires further clarification. The possibility exists that C57BL mice did not learn the alcohol aversion well because of the strong
57
preference for alcohol normally shown by this strain. This explanation is a plausible alternative to the hypothesis that the insensitivity to alcohol or lack of salience was responsible for the learning deficiency, especially since it has been found that levels of pretest intake are related to the amount of learned aversion [2]. The fact that both strains did learn to avoid a presumably highly preferred saccharin solution would seem to argue against this explanation based on initial preference. However, it is also true that the overall amount of aversion shown to saccharin was not very great and it is also remotely conceivable that C57BL mice have a higher preference for the alcohol than for the saccharin. With rats, it has been shown that palatability is not a critical variable in determining learned aversions [7] and that rats readily do learn aversions to highly palatable sucrose or saccharin solutions [13]. Experiment 3 was performed to clarify whether with procedural changes these mouse strains would show strong learned aversions to solutions which are known to be highly preferred.
EXPERIMENT 3
Two specific changes were introduced in Experiment 3; one was the use of a highly palatable 15 per cent sucrose solution instead of the saccharin solution used in Experiment 2. This change was made because it was important to test the ability of C57BL mice to learn an aversion to a solution which they demonstrably prefer and it has been shown that C57BL mice have a very strong preference for 15 per cent sucrose over alcohol [16]. The second change was a change in the testing procedure. In Experiment 2 all mice had lived and were tested in their home cages in which food pellets were always available. When the 10 rain tests were given, whether with water, alcohol, or saccharin, the mice would typically drink for several minutes and then start eating. It is likely that this procedure interfered with the salience of the drinking stimulus since the mice in effect were injected with LiC1 after a combination of drinking and eating. In Experiment 3, mice lived in group cages where food was always available but were tested in separate individual cages in which no food was present.
Method The animals were 7 male C57BL/6 and 7 male BALB/c mice, about 17 weeks old, which were from the same shipment as the mice of Experiment 2. The testing procedure was the same as in Experiment 2 except that no control groups were used and the animals were moved for the drinking tests to individual cages in which no food was present. F o r the first three days, Days 1-3, the mice were given a 1 hr daily water test. On D a y 4, the treatment day, all animals received 6.7 per cent v/v ethyl alcohol for I0 rain and 3 min later were removed from the test cages and injected intraperitoneally with 0.02 ml per g body weight of 0.15 m LiCl. On Days 5 and 6, the mice received 1 hr water daily and on D a y 7, they were tested for 10 min with the alcohol solution. The mice were kept on ad lib food and water for 14 days and then an identical 7 day testing procedure was conducted using the same 7 mice of each strain but with 15 per cent sucrose (w/v) as the test stimulus instead of alcohol. These days were called experimental Days 8-14 with the sucrose tests occurring on Days 11 and 14.
NACHMAN, LARUE AND 11! MAGNI!N
58 Results and Discussion Figure 5A contains the amount of alcohol drunk by each strain on Day 4, before the LiCI treatment, and on the test day, Day 7; Fig. 5B contains the comparable data for sucrose intakes of each strain. The alcohol conditioning results were similar to those of Experiment 2 in showing a much greater learned aversion to alcohol by BALB/c mice than by C57BL mice. While the BALB/c mice decreased their intake from 0.81 ml to 0.11 ml (p < 0.001), the C57BL group had a relatively small but significant decrease in alcohol intake from 1.04 ml to 0.76 ml (p < 0.05). The decrease in the alcohol intake of BALB/c mice was significantly greater than that shown by the C57BL mice (p .~ 0.01). ru~ 150
100.
050
[~e Post
Pre Post
Pre Post
Pre Post
BALB/(:
C 57 BL
BALB/¢
C 57 BL
ALCOHOl
@SUCROSE
FIG. 5. Mean ( ± S.E.) amount (ml) of alcohol (A) and of sucrose (B) drunk on the treatment day, before the LiCI treatment (Pre), and on the test day (Post), for experimental mice of each strain. In contrast to the alcohol results, there were no significant differences between the strains when sucrose was used as the test stimulus and both strains drank only small amounts of sucrose on the test day. For each strain the decrease in sucrose intake following the LiCI treatment was highly significant (ps < 0.001) indicating that both strains were capable of learning a strong aversion even when a highly preferred solution was used.
G E N E R A l . DISCUSSION
Experiment 1 demonstrates that the aversion for alcohol exhibited by BALB/c mice is a response to the olfactory stimulating property of alcohol solutions while the preference exhibited by the C57BL strain is not. In addition, Experiments 2 and 3 suggest that the two strains differ in their responsiveness to the sensory stimulating properties of alcohol. Thus, while metabolic differences with regard to alcohol may also exist in the two strains, it is clear that the strain differences in alcohol preference behavior cannot be attributed exclusively to these metabolic differences. The fact that an alcohol aversion appears to be immediately exhibited by naive BALB/c mice, and does not recur sometime after its elimination by the olfactory bulb lesions, strongly supports the idea that the alcohol aversion of this strain is a response to the odor of alcohol and is not dependent on a particular metabolic effect of alcohol. It is less clear why C57BL mice prefer alcohol to water and why BALB/c mice do not develop the same preference after the elimination of olfactory stimulation. A possible explanation is that the preference is based on gustatory or other sensory cues and that these cues are lacking or less prominent in BALB/c mice. It is also possible that in C57BL mice, but not in BALB/c mice, alcohol exerts a positive metabolic effect which serves to induce the preference. It is evident that in order to be able to show a consistent alcohol preference each day, even when bottle positions are changed, C57BL mice must be responding to some type of sensory stimulus, such as the taste of the alcohol. However, the sensory stimulus could be operating either to produce a preference directly or as a cue to mediate learning induced by positive metabolic effects. It is not known which of these is the case or whether both may be operating. The fact that the C57BL's preference for alcohol seems to increase with experience is suggestive evidence that post-ingestional factors may play an important role in the alcohol preference of C57BL mice, Thus, while strain differences in alcohol aversion appear to be dependent on sensory factors and are specific to the olfactory system, the existence of strain differences in alcohol preferences may be more dependent on the possible reinforcing effects of postingestional factors.
REFERENCES 1. Amit, Z. and M. H. Stern. Alcohol ingestion without oropharyngeal sensations. Psychonom. Sci. 15: 162-I63, 1969. 2. Bauermeister, J. J. and R. W. Sehaeffer. Relations between preconditioned rate of solution ingestion and rate of postirradiation intake. PhysioL Behav. 4: 1019-1021, 1969. 3. Forsander, O. A. Metabolic tolerance to alcohol as a possible limiting factor in its consumption. Q. Jl Stud. Alcohol 23: 480--482, 1962. 4. Fuller, J. L. Measurement of alcohol preference in genetic experiments. J. comp. physiol, Psychol. 57: 85-88, t964. 6. Kakihana R., D. R. Brown, G. E. MeClearn and I. R. Tabershaw. Brain sensitivity to alcohol in inbred mouse strains. Science 154: 1574-1575, 1966. 7. Kalat, J. W. and P. Rozin. Salience. A factor which can override temporal contiguity in taste-aversion learning. J. comp. physiol. Psychol. 71: 192-197, 1970. 8. LeMagnen, J. and P. Marfaing-Jallat. Le r61e des aff6rences buccales clans le det6rminism¢ des consommations spontan~es d'alcool chez ie rat. J. Physiol. Paris, 53: 407--408, 1961.
9. MeClearn, G. E. and D. A. Rodgers. Differences m alcohol preference among inbred strains of mice. Q. Jl Stud. Alcohol. 20: 691--695, 1959. 10. McClearn, G. E. and D. A. Rodgers. Genetic factors in alcohol preference of laboratory mice. J. comp. physiol. Psychol. 54: 116-119, 1961. 11. Nachman, M., D. Lester and J. LeMagnen. Alcohol aversion in the rat: Behavioral assessment of noxious drug effects. Science 168: 1244-1246, 1970. 12. Peacock, L. J. and J. A. Watson. Radiation-induced aversion to alcohol. Science 143: 1462-1463. 1964. 13. Revusky, S. H. and J. Garcia. Learned aversion over long delays. In: The Psychology of Learning and Motivation: Advances in Research and Theory, 4: edited by G. H. Bower and J. T. Spence. New York: Academic Press, 1970, pp. 1-83. 14. Rodgers, D. A. Factors underlying differences in alcohol preference among inbred strains of mice. Psychosom. Med. 28: 498-513, 1966.
OLFACTORY, OROSENSORY FACTORS A N D ALCOHOL PREFERENCE 15. Rodgers, D. A. and G. E. McCleam. Alcohol preference of mice. In: Roots of Behavior, edited by E. L. Bliss. New York: Hoeber-Harper, 1962. 16. Rodgers, D. A. and G. E. McClearn. Sucrose versus ethanol appetite in inbred strains of mice. Q. Jl Stud. Alcohol. 25: 26-35, 1964. 17. Rodgers, D. A., G. E. McClearn, E. L. Bennett and M. Hebert. Alcohol preference as a function of its caloric utility in mice. J. comp. physiol. Psychol. 56: 666-672, 1963.
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18. Thomas, K. Selection and avoidance of alcohol solutions by two strains of inbred mice and derived generations. Q. Jl Stud. Alcohol. 30: 849--861, 1969. 19. Whitney, G. D. and Y. Whitney. Ethanol toxicity in the mouse and its relationship to ethanol selection. Q. Jl Stud. Alcohol. 29:~4 48, 1968.
The Role of Olfactory and Orosensory Factors in the Alcohol Preference of Inbred Strains of Mice' MARVIN
NACHMAN
Department of Psychology, University of California, Riverside, California, U.S.A. AND CHR1STIANE LARUE AND JACQUES LE MAGNEN
Laboratoire de Neurophysiologie, Colldge de France, Paris, France (Received 7 July 1970)
NACHMAN,M., C. LARUE AND J. LE MAGNEN. The role of olfactory and orosensory factors in the alcohol preference
of inbred strains of mice. PHYSIOL. BEHAV. 6 (1) 53--59, 1971.--Removal of the olfactory bulbs eliminated the aversion to alcohol which is seen in normal BALB/c mice but did not abolish the preference for alcohol seen in normal C57BL mice. This result, along with the fact that BALB/c mice appear to avoid alcohol immediately, without prior experience, led to the hypothesis that BALB/c mice are more responsive than are C57BL mice to alcohol as a sensory stimulus. To test this hypothesis, a conditioning procedure was used in which normal animals of both strains drank alcohol in a single bottle test and were then injected with toxic lithium chloride. It was found that BALB/c mice learned aversions to alcohol, saccharin, and sucrose, whereas C57BL mice learned aversions to saccharin and sucrose but were deficient in learning an alcohol aversion. It was concluded that BALB/c mice normally avoid alcohol because of its odor but that for C57BL mice, other sensory cues or post-ingestional factors play an important role in their preference for alcohol. Olfaction Orosensory factors Alcohol preference Mouse strains Olfactory bulbs factors Taste preference Learned aversions Conditioned taste aversions
Post-ingestional
significant role in strain differences in alcohol selection. Perhaps most important is the fact that the strain differences can be seen on the first day of testing with the solutions, a finding which suggests that these differences may exist before learning due to post-ingestive factors has occurred. The role of taste in alcohol intake is further indicated by the fact that alcohol intake in mice can be increased by sweetening the solutions with sucrose [16] and that in rats, a positive correlation exists between quinine aversion thresholds and alcohol intake [8]. The specific influence of olfaction in alcohol selection has also been demonstrated by the finding that bulbectomized rats, in comparison with normal rats, show less of an alcohol preference at low alcohol concentrations and less of an alcohol aversion at high concentrations [5]. Elimination of oropharyngeal sensations by directly infusing the alcohol into the stomach has also been shown to result in increased alcohol intake [1]. The present experiments used the high alcohol preferring strain, CB57BL, and the low alcohol preferring strain, BALB/c, and the first experiment was designed to determine
INBRED strains of mice differ greatly in the degree to which they will ingest alcohol. When given an ad lib choice between 10% v/v alcohol and water, for example, C57BL mice will show a strong preference for the alcohol solution whereas the BALB/c or DBA/2 strains will ingest very little alcohol [9, 101. While it is evident that these strain differences in alcohol intake are genetically determined, it is not known what physiological differences in the strains are responsible for the differences in alcohol selection. Self-selection of foods and fluids is often determined both by sensory factors and by postabsorptive effects. In the case of alcohol selection, there has been considerable research attempting to relate the strain differences in alcohol intake to strain differences in alcohol metabolism or toxicity [6, 17, 19]. However, there has been very little work reported which has attempted to relate strain differences in alcohol intake to possible gustatory or olfactory factors. Several lines of evidence, although each inconclusive, are suggestive of the hypothesis that sensory factors may play a
1This research was supported in part by NIH Special Research Fellowship (to M.N.) 7 F03 MH14939-01A1 and was conducted at the Coll6ge de France. 53
54
NACttMAN, LARUE ANt) IL MAGNI:N
whether removal of the olfactory bulbs would effect the strain differences in alcohol intake which normally exists in these mice. EXPERIMENT
1
Method The mouse strains were obtained from the animal laboratory of Orl~ns-La Source, France. The animals were 30 male C57BL/6 and 30 male BA_LB/c mice, which ranged in age from 8 to 13 weeks when testing began. The olfactory bulbs were bilaterally removed, under nembutal anesthesia, for 15 mice of each strain, while the other 15 mice of each strain served as unoperated controls. The bulbectomies were performed with suction and care was taken to entirely remove the olfactory bulbs. After a one week operative recovery period, all mice were given an ad lib two-bottle choice between a 10% v/v ethyl alcohol solution and tap water. The mice were caged individually in cages 16 × 16 × 20 cm and food pellets were always available. The test bottles used were 25 ml graduated cylinders with stainless steel spouts protruding 4 cm into the cage and separated by about 4 cm. The twobottle choice test was carried out for 12 days with the amount ingested measured daily; no attempt was made to correct for evaporation or drippage which was estimated to be relatively little, on the order of 0.1-0.2 ml per day. The bottles were refilled and their positions switched every 3 days. Alcohol preference scores were obtained daily and expressed as a percentage by dividing the alcohol intake by the total fluid intake and multiplying by 100. For each mouse, the alcohol containing bottle and stainless steel spout and the water bottle and stainless steel spout were kept constant throughout the 12 days. After this test period, stainless steel spouts were switched on several occasions to determine whether a particular spout rather than the test solution might be determining the preference. In the case of one mouse, a BALB/c bulbectomized, it was evident that the animal's large alcohol intake was determined by differences in the flow of the two spouts and the data of this animal were discarded. In addition, one C57BL bulbectomized mouse died during the experiment leaving 14 bulbectomized and 15 controls of each strain. As will be seen in the results below, the data of the first 12 days of testing indicated that the BALB/c controls showed the typical alcohol aversion which other investigators have previously reported, but that the bulbectomized BALB/c mice did not show any apparent aversion to alcohol. These results were found using a standard procedure of changing the position of bottles every 3 days, however, and this may have made it difficult for the bulbectomized BALB/c mice to locate the position of the alcohol and water bottles. Also, these results were found in the first 12 days of testing of bulbectomized and normal mice which had not previously had experience with alcohol solutions. To examine whether these results were limited to these test conditions, additional alcohol vs. water testing was done with less changing of bottle positions and using bulbectomized BALB/c mice which had previously shown an aversion to alcohol when they had been tested as normals. Eight of the control BALB/c mice, randomly selected from those which had shown a clear aversion to alcohol in the tests of the first 12 days, were bilaterally bulbectomized after they had had 21 days of alcohol vs. water testing. They were maintained, without interruption, for an additional 12 days of alcohol vs. water choice in which the bottles were changed
every 3 days and then tested for 24 days during which the position of the bottles was changed only once, after 12 days. For comparison, 6 of the BALB/c mice which had been bulbectomized before any testing, were randomly selected, maintained for the same total of 33 days of alcohol vs. water choice with the bottle positions changed every 3 days, and then also tested for 24 days of alcohol vs. water choice with bottle positions changed once after 12 days. In addition, 14 C57BL mice were tested in exactly the same way as the 14 BALB/c mice, with 8 of the C57BL mice having been bulbectomized after first serving as controls and the remaining 6 C57BL mice serving as bulbectomized from the beginning of the experiment. Because of drainage of one bottle, data were recorded from 14 BALB/c and 13 C57BL mice during the 24-day test. At the end of testing, the mice were sacrificed and the brains examined for the extent of the olfactory bulb ablations. A sagittat brain section of a normal and of a bulbectomized BALB/c mouse is presented in Fig. 1. Results The mean of the daily alcohol preference scores for each mouse for the first 12 day test period is presented in Fig. 2. For the C57BL strain, removal of the olfactory bulbs had no apparent effect on the overall 12-day preference for alcohol; the mean alcohol preference score was 73.8 per cent for the C57BL controls and 76.2 per cent for the C57BL bulbectomized mice. In contrast, the mean alcohol preference score for the BALB/c controls was 17.9 per cent which was significantly less than the mean score of 47.5 per cent for the BALB/c bulbectomized group (p < 0.01, all ps reported in Experiment 1 are for Mann-Whitney U-tests). The difference in alcohol intake between BALB/c controls and BALB/c bulbectomized mice was clearly evident on the very first day of testing. On this day, the BALB/c controls showed a strong alcohol aversion with 14 of the 15 mice drinking less alcohol than water and the mean intake for all 15 was 0.66 ml of alcohol compared to 3.51 ml of water. A further way of indicating the very small amounts of alcohol taken by BALB/c controls on the first day is to examine the
3 Control
0
......
n
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........
BAL 8/c
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r-n.
i 0
,..,F1
......
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FIG. 2. Alcohol preference scores of individual control and bulbectomized mice of each strain during the first 12 days of two-bottle alcohol vs. water preference tests.
~.........
!ii! ¸ ii! iiiii~iiiiii
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FIG. 1. Unstained sagittal sections of a normal mouse brain (A) and of a brain from which the olfactory bulbs had been removed (B).
(facing page
54)
OLFACTORY, OROSENSORY FACTORS AND ALCOHOL PREFERENCE scores of individual animals. Of the 15 BALB/c controls, 13 drank 0.09 ml or less of alcohol and of these, 7 drank 0.4 ml or less of alcohol. Considering that spillage and evaporation probably accounted for about 0.1 or 0.2 ml of these amounts, it can be seen that BALB/c control mice ingest very little alcohol when first exposed to it in a choice situation. In contrast, the BALB/c bulbectomized mice showed no evidence of an alcohol aversion on the first day, the mean intake of this group on the first day was 1.7 ml of alcohol and 1.3 ml of water and the alcohol preference scores of all mice were distributed throughout the range between 21 and 81 per cent alcohol preference. The first day intake scores of the C57BL mice also revealed a significant difference between the controls and the bulbectomized (p < 0.05). In this case, however, the controls appeared to be relatively indifferent to alcohol with a mean preference score of 50.9 per cent and only 8 of the 15 control mice drank more alcohol than water, while in comparison, the bulbectomized C57BL mice had a mean preference score of 72.0 per cent on the first day and 13 of the 14 mice drank more alcohol than water. The control C57BL mice progressively increased their alcohol preference during the first few days to equal that of the bulbectomized C57BL mice. F o r example, on the second, third and fourth days of testing, the number of control C57BL mice which drank more alcohol than water increased to 9, 12 and 14 animals, respectively. The data of McClearn and Rogers [9] also indicate that C57BL mice do not show an alcohol preference on the first day and Rodgers [14] states that initial establishment of stabilized preferences in mice requires 3 or 4 days. Figure 3 contains the alcohol preference scores of the bulbectomized mice of each strain which were tested for 24 days with the bottle positions changed only once. These results are essentially the same as those seen for the bulbectomized mice in Fig. 2, i.e. the bulbectomized C57BL mice show a clear alcohol preference, with a mean alcohol preference score of 81.9 per cent and the bulbectomized BALB/c mice drink approximately equal amounts of alcohol and water, with a mean alcohol preference score of 55.9 per cent. Thus, the fact that the animals were more experienced by the time of the 24-day tests or that bottle positions were changed less often had no apparent influence on the alcohol preference scores. Also, there was no evidence of any trend within each 12-day segment. Figure 3 contains both the data of animals which had been bulbectomized at the beginning of the experiment as well as those which had first served as controls
C 57 BL/6
32]
rl " ~b
io
AlCOhOl
30
~
preference
" fro " 6b
I percent
" 70
" 8o
of total
ab
t~0
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FIG. 3. Alcohol preference scores of individual bulbectomized mice of each strain during 24-day tests in which bottle positions were changed once after 12 days. The shaded portions represent animals which had been tested before and after they were bulbectomized and the unshaded portions represent animals tested only after bulbectomy.
55
and it was found that these subgroups did not differ in their alcohol preference scores (.as > 0.05). In the case of the BALB/c mice, this indicates that the lack of aversion to alcohol following the removal of the olfactory bulbs was not influenced by whether or not the mice had previously had experience with avoiding alcohol.
Discussion It is likely that the effects of the bulbectomy operations in this study were due specifically to the removal of the olfactory bulbs. Rodgers and McClearn in a review article [15] cite experiments in which removal of only the olfactory bulbs produced inconclusive effects on the alcohol aversion of mice of the A strain but that removal of the anterior third of the cerebrum including the olfactory bulbs produced increased alcohol intake in both A and BALB/c mice. In the present study, frontal damage was typically slight, as illustrated in Fig. 1. The lesions in general were restricted to the olfactory bulbs and there was no evidence of a relationship between alcohol preference scores and extent of lesion. The alcohol preference scores indicate that olfactory mechanisms play an important role in the normal avoidance of alcohol by the BALB/c strain. The bulbectomized BALB/c mice appear to be relatively indifferent to alcohol as judged by the fact that as a group they drank approximately equal amounts of water and alcohol and all animals ranged between alcohol preference scores of 26.4 and 77.9 per cent. However, although the animals did not show strong aversions, they apparently could discriminate between the alcohol and water bottles as revealed by the day to day intake scores which show that some animals have a small but consistent daily preference for either the alcohol solution or the water. Furthermore, the evidence indicates that the lack of aversion to alcohol by the bulbectomized BALB/c mice is not due to an inability to locate the position of the alcohol bottle since the BALB/c bulbectomized mice still did not show any sign of avoiding alcohol when bottle positions were kept constant for 12 days, even if the animals had previously had experience with avoiding alcohol when they were controls. On the basis of these results, it seems justifiable to conclude that the BALB/c mice normally avoid 10 per cent alcohol because of the odor of this substance and that in the absence of the sense of smell, the amount of alcohol ingested does not have aversion producing effects. This conclusion differs, of course, from those which relate an animal's limited alcohol intake to a metabolic tolerance factor [3] or to the capacity to metabolize alcohol as shown by acetaldehyde levels or by limiting amounts of liver alcohol dehydrogenase enzyme [14]. While it is possible that both olfactory and metabolic factors may be operating, the present results suggest that metabolic factors are not principally responsible for limiting the intake of alcohol in the BALB/c mice. If the mice were avoiding alcohol because of some toxic metabolic consequence, it would be difficult to understand why removal of the olfactory bulbs would permit the animals to ingest larger amounts of alcohol without producing an aversion due to the same toxic consequences. The small amounts of alcohol ingested by BALB/c mice on the first day of testing provides further support for the idea that sensory factors rather than metabolic factors are directly responsible for the observed alcohol aversion. Although it is certainly possible that a learned aversion could have occurred within the first test day, the amounts of alcohol ingested were extremely small, and were much less than estimates of the amounts that can be metabolized in 24 hr [16]. These results
56
N A C t t M A N . LAR[)[; A N I ) 11
suggest that the animals were responding directly to the smell or taste of the alcohol rather than avoiding it because of some consequence of intake. It should be noted, however, that the possible existence of strain differences in sensory response to alcohol in no way negates or argues against the concomitant existence of strain differences in metabolic capacity. In fact, it may be that sensory differences have evolved because of their adaptive value in relation to metabolic differences. The C57BL mice differ in several important ways from BALB/c mice in their intake of alcohol and it is likely that differing physiological substrates may be involved in influencing alcohol intake in the two strains, a suggestion which has arisen as a result of genetic analysis of the strains [4]. The most obvious difference between the strains is that BALB/c mice avoid alcohol, while the C57BL mice show a strong preference for alcohol over water. Second, the alcohol aversion shown by BALB/c mice depends on the olfactory bulbs whereas the preference for alcohol by C57BL mice is maintained after removal of the olfactory bulbs. Third, the aversion to alcohol shown by the BALB/c mice seems to be immediate and apparently does not have to be learned whereas the preference for alcohol by C57BL mice is not seen immediately and may take several days to develop. Fourth, it is likely that C57BL mice have a much higher threshold for preferring alcohol than BALB/c mice have for avoiding alcohol, since large differences in these thresholds have been reported between C57BL mice and another alcohol avoiding strain, DBA/2 [18]. The fact that BALB/c mice show an immediate aversion to alcohol and this aversion is eliminated by bulbectomy suggests that the animals are responding to the odor of alcohol as an aversive stimulus. In contrast, the high threshold at which C57BL mice first select alcohol, along with the fact that the alcohol preference remains after bulbectomy, suggests that C57BL mice are either relatively insensitive to the odor of alcohol or that if sensitive, the odor is much less noxious and can be readily overcome by some positively reinforcing aspects of the alcohol. An examination of the first day alcohol preference scores in normal and bulbectomized C57BL mice gives some support for the idea that C57BL mice may have a slight negative response to the odor of alcohol. Whereas the normal C57BL mice showed no preference for alcohol over water on the first day and gradually increased their preference over days, the bulbectomized C57BL mice preferred alcohol from the first day. Also, the first day data of McClearn and Rodgers [9] indicates a small aversion to alcohol by normal female C57BL mice which later becomes a preference.
~IA(iNI N
solution was used because it was believed that a It) per cent solution might be strong enough for both strains to learn the aversion and thus obscure the difference between the strains. Method The animals were 30 male C57BL and 30 male BALB/c mice ranging in age from 7 to 14 weeks and obtained from the same supplier as in Experi,nent 1. They were housed in individual cages with food pellets always available. For each strain, mice were assigned randomly to an experimental grot,p (n 20) or to a control group (n := I0). The testing procedure was modified slightly from one previously used with rats to produce learned alcohol aversions [11]. All animals were given a single water bottle in their home cage for 1 hr daily for 3 days and the amount ingested in the first 10 min was measured. On the 4th day, the treatment day, all animals were given a single-bottle 10-min test with 6.7 per cent v/v ethyl alcohol. Three rain after the alcohol test, the experimental groups of each strain were injected intraperitoneally with 0.02 ml per g body weight of 0.15 m LiCI, while the control animals were not treated. The time interval of 3 rain was chosen arbitrarily although it was known to be well within the effective times to produce learned aversions in rats. On the two successive days, Days 5 and 6, the animals were given access to water for one hour to extinguish any generalized aversion to drinking and on Day 7, the test day, they were again given a single-bottle 10-rain test wilh 6.7 per cent ethyl alcohol and their intake recorded. The results showed that the experimental groups of the two strains did differ in the degree to which they avoided alcohol on the test day. To determine whether this strain difference could be attributed to the alcohol stimulus or whether a similar strain difference would occur with other substances, the same experimental procedure was then repeated in a second part of the experiment using 0.I per cent saccharin solution as the conditioned stimulus instead of alcohol. The same animals were retested as experimental and control animals and the second part of the experiment was started between 10 and 30 days after the end of the first part. During this time interval the animals were kept on ad lib food and water. The sequence of testing in the second part of the experiment was identical to that of the first part: on the first three days, experimental Days 8-10, the mice received 1 hi" water daily; on Day 11, they received 0.t per cent sodium saccharin for 10 min and 3 rain later the experimental groups were injected with LiCt; on Days 12 and 13 the mice received 1 hr water daily and on Day 14 they were ~ested for I0 rain with 0.1 per cent saccharin.
EXPERIMENT 2
One of the conclusions of Experiment 1 was that C57BL mice may be less responsive than BALB/c mice to sensory stimulating properties of alcohol. Experiment 2 was designed to further test this conclusion by using a conditioning procedure in which the ingestion of alcohol was followed by a toxic injection of lithium chloride and the animals subsequently tested for their learned aversion to ingesting alcohol. It was hypothesized that BALB/c mice would readily learn to avoid ingesting alcohol but that C57BL mice would be relatively deficient in such learning. Similar conditioned aversions to ingesting alcohol have been reported in rats [11] and in mice [12], and it has also shown that the salience of an ingested solution is an important predictor of taste-aversion learning [7]. In Experiment 2, a 6.7 per cent v/v ethanol
Results and Discussion Figure 4A contains the amount of alcohol drunk on the test day, Day 7, for the experimental and control groups of each strain. It can be seen that the BALB/c mice had learned the alcohol aversion since the 0.48 ml drunk by the experimental group was significantly less than the 1.I 3 ml drunk by their controls (p < 0.001, all ps reported in Experiments 2 and 3 are for two-tailed t tests). In contrast, the 0,86 ml of alcohol drunk by the experimental C57BL mice, while slightly less, was not significantly different from tbel 0.98 ml alcohol drunk by their controls (p > 0.2). On Day 4, before the LiCI treatment, the BALB/c mice showed no evidence of an alcohol aversion. The alcohol intake of BALB/c mice was 1.24 ml which was not significantly
OLFACTORY, OROSENSORY FACTORS AND ALCOHOL PREFERENCE
ml 2,00. ~
control experimental
1.50 1,00 0,50.
BALB/c
C 57BL
ALCOHOL
BALB/c
C 57 BL
(~SACCHARIN
FIG. 4. Mean (~: S.E.) amount (ml) of alcohol (A) and of saccharin (B) drunk on the test day by control and experimental mice of each strain.
different (p > 0.2) than the 1.08 ml alcohol intake of C57BL mice and in both strains, the amount of alcohol ingested on Day 4 was almost identical with the amount of water ingested in 10 min on Day 3. This was probably due to the use of a single-bottle forced-choice alcohol test as well as to the weaker 6.7 per cent alcohol used. F o r both strains, there were no significant differences in alcohol intake of control animals between Day 4 and Day 7 (ps > 0.2). The alcohol conditioning procedure did not have any apparent effect on the saccharin intake scores of Day 11 ; on this day, there were no significant differences between experimental and control mice of either strain in saccharin intake (ps > 0.1). Figure 4B contains the saccharin intake scores for both strains on the test day, D a y 14. In contrast to the results with alcohol, it can be seen that the C57BL mice learned the saccharin aversion as well as, if not somewhat better than did the BALB/c mice. The experimental BALB/c mice drank 1.29 ml of saccharin on Day 14 which was significantly less than the 1.72 ml drunk by their controls (p < 0.05) and the experimental C57BL mice drank 0.87 ml which was much less than the 1.76 ml drunk by their controls (p < 0.001). On Days 5 and 12, the days immediately following each of the LiCI treatments, the 10-min water intake of the experimental animals of each strain was about 15 per cent less than the controls. This decrease in water intake reflected either a generalized aversion to drinking or a residual effect of the LiCI sickness. However, by the next day in each case, Day 6 and Day 13, there was no longer a difference between the water intake of the experimentals and controls of either strain in the first 10 min or in the 60 min totals. Thus, the aversions shown to alcohol on D a y 7 and to saccharin on D a y 14 could not be attributed to any residual sickness from the LiCI but were specific to the ingested solutions. The alcohol conditioning results of Experiment 2 showed that C57BL mice did not learn an alcohol aversion as well as BALB/c mice. The saccharin tests, by finding that both groups did learn a saccharin aversion, demonstrated that the strain differences in learned alcohol aversion was not due to some extraneous variable such as learning ability or sensitivity to LiCI but was specific to alcohol. However, one aspect of the Experiment 2 results requires further clarification. The possibility exists that C57BL mice did not learn the alcohol aversion well because of the strong
57
preference for alcohol normally shown by this strain. This explanation is a plausible alternative to the hypothesis that the insensitivity to alcohol or lack of salience was responsible for the learning deficiency, especially since it has been found that levels of pretest intake are related to the amount of learned aversion [2]. The fact that both strains did learn to avoid a presumably highly preferred saccharin solution would seem to argue against this explanation based on initial preference. However, it is also true that the overall amount of aversion shown to saccharin was not very great and it is also remotely conceivable that C57BL mice have a higher preference for the alcohol than for the saccharin. With rats, it has been shown that palatability is not a critical variable in determining learned aversions [7] and that rats readily do learn aversions to highly palatable sucrose or saccharin solutions [13]. Experiment 3 was performed to clarify whether with procedural changes these mouse strains would show strong learned aversions to solutions which are known to be highly preferred.
EXPERIMENT 3
Two specific changes were introduced in Experiment 3; one was the use of a highly palatable 15 per cent sucrose solution instead of the saccharin solution used in Experiment 2. This change was made because it was important to test the ability of C57BL mice to learn an aversion to a solution which they demonstrably prefer and it has been shown that C57BL mice have a very strong preference for 15 per cent sucrose over alcohol [16]. The second change was a change in the testing procedure. In Experiment 2 all mice had lived and were tested in their home cages in which food pellets were always available. When the 10 rain tests were given, whether with water, alcohol, or saccharin, the mice would typically drink for several minutes and then start eating. It is likely that this procedure interfered with the salience of the drinking stimulus since the mice in effect were injected with LiC1 after a combination of drinking and eating. In Experiment 3, mice lived in group cages where food was always available but were tested in separate individual cages in which no food was present.
Method The animals were 7 male C57BL/6 and 7 male BALB/c mice, about 17 weeks old, which were from the same shipment as the mice of Experiment 2. The testing procedure was the same as in Experiment 2 except that no control groups were used and the animals were moved for the drinking tests to individual cages in which no food was present. F o r the first three days, Days 1-3, the mice were given a 1 hr daily water test. On D a y 4, the treatment day, all animals received 6.7 per cent v/v ethyl alcohol for I0 rain and 3 min later were removed from the test cages and injected intraperitoneally with 0.02 ml per g body weight of 0.15 m LiCl. On Days 5 and 6, the mice received 1 hr water daily and on D a y 7, they were tested for 10 min with the alcohol solution. The mice were kept on ad lib food and water for 14 days and then an identical 7 day testing procedure was conducted using the same 7 mice of each strain but with 15 per cent sucrose (w/v) as the test stimulus instead of alcohol. These days were called experimental Days 8-14 with the sucrose tests occurring on Days 11 and 14.
NACHMAN, LARUE AND 11! MAGNI!N
58 Results and Discussion Figure 5A contains the amount of alcohol drunk by each strain on Day 4, before the LiCI treatment, and on the test day, Day 7; Fig. 5B contains the comparable data for sucrose intakes of each strain. The alcohol conditioning results were similar to those of Experiment 2 in showing a much greater learned aversion to alcohol by BALB/c mice than by C57BL mice. While the BALB/c mice decreased their intake from 0.81 ml to 0.11 ml (p < 0.001), the C57BL group had a relatively small but significant decrease in alcohol intake from 1.04 ml to 0.76 ml (p < 0.05). The decrease in the alcohol intake of BALB/c mice was significantly greater than that shown by the C57BL mice (p .~ 0.01). ru~ 150
100.
050
[~e Post
Pre Post
Pre Post
Pre Post
BALB/(:
C 57 BL
BALB/¢
C 57 BL
ALCOHOl
@SUCROSE
FIG. 5. Mean ( ± S.E.) amount (ml) of alcohol (A) and of sucrose (B) drunk on the treatment day, before the LiCI treatment (Pre), and on the test day (Post), for experimental mice of each strain. In contrast to the alcohol results, there were no significant differences between the strains when sucrose was used as the test stimulus and both strains drank only small amounts of sucrose on the test day. For each strain the decrease in sucrose intake following the LiCI treatment was highly significant (ps < 0.001) indicating that both strains were capable of learning a strong aversion even when a highly preferred solution was used.
G E N E R A l . DISCUSSION
Experiment 1 demonstrates that the aversion for alcohol exhibited by BALB/c mice is a response to the olfactory stimulating property of alcohol solutions while the preference exhibited by the C57BL strain is not. In addition, Experiments 2 and 3 suggest that the two strains differ in their responsiveness to the sensory stimulating properties of alcohol. Thus, while metabolic differences with regard to alcohol may also exist in the two strains, it is clear that the strain differences in alcohol preference behavior cannot be attributed exclusively to these metabolic differences. The fact that an alcohol aversion appears to be immediately exhibited by naive BALB/c mice, and does not recur sometime after its elimination by the olfactory bulb lesions, strongly supports the idea that the alcohol aversion of this strain is a response to the odor of alcohol and is not dependent on a particular metabolic effect of alcohol. It is less clear why C57BL mice prefer alcohol to water and why BALB/c mice do not develop the same preference after the elimination of olfactory stimulation. A possible explanation is that the preference is based on gustatory or other sensory cues and that these cues are lacking or less prominent in BALB/c mice. It is also possible that in C57BL mice, but not in BALB/c mice, alcohol exerts a positive metabolic effect which serves to induce the preference. It is evident that in order to be able to show a consistent alcohol preference each day, even when bottle positions are changed, C57BL mice must be responding to some type of sensory stimulus, such as the taste of the alcohol. However, the sensory stimulus could be operating either to produce a preference directly or as a cue to mediate learning induced by positive metabolic effects. It is not known which of these is the case or whether both may be operating. The fact that the C57BL's preference for alcohol seems to increase with experience is suggestive evidence that post-ingestional factors may play an important role in the alcohol preference of C57BL mice, Thus, while strain differences in alcohol aversion appear to be dependent on sensory factors and are specific to the olfactory system, the existence of strain differences in alcohol preferences may be more dependent on the possible reinforcing effects of postingestional factors.
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