Lateralization of learned taste aversion by cortical spreading depression

Physiology and Behavior, Vol. 10, pp. 79-83, Brain Research Publications Inc., 1973. Printed in U.S.A.

Lateralization of Learned Taste Aversion by Cortical Spreading Depression PATRICIA P. LEHR AND MARVIN NACHMAN i

Department o f Psychology, University o f California Riverside, California 92502

(Received 11 July 1972)

I FHR, P. P. AND M. NACHMAN. Lateralization of learned taste aversion by cortical spreading depression. PHYSIOL. 10(1) 79-83, 1973.-Male Sprague-Dawley rats which were taught a strong aversion to ingestion of lithium chloride under unilateral cortical spreading depression failed to show this aversion when spreading depression was changed to the opposite hemisphere. Control rats which learned the aversion with both hemispheres functional show a very strong aversion to LiCI in subsequent tests with either both hemispheres functional or one hemisphere under spreading depression. These results indicate that cortical participation is important for learning of a taste aversion. BEHAV.

Cortical spreading depression

Learned taste aversion

LiCI

CORTICAL spreading depression (CSD) elicited by topical application of KCI has been used extensively in the study of interhemispheric transfer of learning. When an animal learns a task with one hemisphere under CSD and is subsequently tested with the other hemisphere under CSD, the animal may or may not show transfer of the learning from the trained hemisphere to the previously depressed hemisphere. The results from a number of studies suggest that interhemispheric transfer may be dependent on the type of task [6]; when the learning task involved is relatively complex, such as in active avoidance and appetitive tasks [ 1, 4, 19, 22, 23 ], the learning is found to be restricted to the hemisphere that is functional during learning. When the learning task involved is relatively less complex, such as in passive avoidance [1, 7, 9, 21], conditioned respiratory response [ 20], and habituation [ 26], the learning appears to be transferred from the functional hemisphere to the nonfunctional hemisphere. A prevalent hypothesis for this task difference is that the less complex tasks are mediated by subcortical mechanisms and that learning is therefore available to both hemispheres. Best and Zuckerman [2] concluded that learned taste aversions were mediated subcortically when they found that the aversion transferred from the trained hemisphere to the previously depressed hemisphere. However, their specific procedure, which used repeated trials of cortical spreading depression, may have had a direct bearing on their results and conclusion.

KC1

Learned taste aversions provide an excellent paradigm to study interhemispheric transfer of learning, particularly because these aversions can be readily produced in a single learning trial. Nachman [15] found that rats became ill within several min after drinking lithium chloride (LiC1) and would henceforth drink very little of it. This one-trial avoidance occurs without exception in normal rats and is retained over long periods of time. Using this training procedure, the present study found that a single trial learned aversion was lateralized to the hemisphere which was functional during training and consequently appears to be dependent on cortical mediation.

EXPERIMENT 1 The first experiment was carried out to determine if rats with one hemisphere under CSD and the other hemisphere functional could learn an aversion to the ingestion of lithium chloride, and if this learned aversion would be restricted to the functional hemisphere.

Method Animals. Eighteen male Holtzman Sprague-Dawley rats weighing 3 0 0 - 3 2 0 g were fed Purina rat chow ad lib and were put on a 23-hr water deprivation schedule throughout the experiment. The animals were divided into 3 groups of

i Requests for reprints should be addressed to : M. Nachman, Department of Psychology, UC, Riverside, California 92502. 79

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LEItR AND NACHMAN

6 rats each, designated by whether the right hemisphere (R). the left hemisphere (L), or neither hemisphere (--) was under CSD during each of 4 tests. These 3 groups were Group R R - - _ Group R R L _ and Group Nonop. Apparatus and Pretraining. The drinking boxes and test procedure have been described previously [ 16]. For 3 days preceding surgery, the rats were given pretraining consisting of a daily 10 min drinking period in the boxes. All rats learned to approach the drinking tubes containing distilled water and to begin drinking as soon as guillotine doors were lifted. At the end of the 10 rain period, the rats were removed from the drinking boxes to their home cages and given 50 rain access to water before again being deprived of water. Surgery. At the end of pretraining, 12 animals were anesthetized with ether, and polyethylene cannulae (PE 240 tubing in 0.5 mm lengths, flared at one end) were implanted bilaterally over the cortex. The flared ends of the cannulae were inserted into trephine holes in the skull just posterior to bregma and approximately 3 mm lateral to the sagittal suture, and were secured with dental acrylic. The tubes were then partially filled with physiological saline. PE 160 polyethylene tubing flared at one end and sealed with dental acrylic served as plugs. Care was taken throughout surgery to keep the dura intact. All animals (the 12 operated and 6 unoperated) were given ad lib water and food for one day following surgery and then were put back on the water deprivation schedule. Training. After 23 hr of water deprivation, a 12-day sequence of daily drinking tests was begun. On the first two days, the rats were given distilled water in the tests and on Day 3, they were presented with 0.12 M LiC1. This 3-day sequence was given 4 times. The interspersed water tests were used to determine whether the animals were drinking normal amounts of water prior to each LiC1 test day. On the first presentation of LiC1 (LiC1 I) on Day 3, 25% KC1 was injected with a syringe into the cannula over the right hemisphere of all 12 operated animals to elicit unilateral CSD. (Pilot studies showed no difference in drinking behavior as to which hemisphere was depressed.) The presence of CSD was ascertained by noting the loss of placing response of the contralateral forepaw within 15 min after KC1 administration. All animals showed this loss of placing and were then given the 10 min drinking test. After each test, the cannulae were flushed with physiological saline before the rats were returned to their home cages for the additional 50 min of water. In order to determine if the animals had learned the aversion in the functional hemisphere, all animals were given a second LiCI test (LiCI II on Day 6) in which the animals under CSD on Day 3 were again depressed on the right side as previously. In the critical test on Day 9 (LiC1 III) 6 animals that had been previously depressed did not undergo CSD (Group RR----), while the other 6 operated animals had KCI injected into the cannula over the previously functional left hemisphere (Group RRL--). Failure by Group R R L - - t o drink LiC1 would mean that the learning was transferred from the right hemisphere to the left hemisphere rather than lateralized to the right hemisphere. On the final LiC1 test (LiCI IV, Day 12), none of the animals were under CSD as a check on whether they had all learned the aversion to LiCI.

Results and Discussion The total amount of liquid each animal drank in each 10

min test was recorded, and tile mean amounts of LiCI consumed per test for each of the 3 groups is given in Table 1. TABLE 1 MEAN AMOUNT(ML) OF LiCI CONSUMED PER 10MINTEST Day 3 LiCI 1

Day 6 LiCI II

Day 9 LiCI !11

Day 12 LiCI IV

RRL--

12.2

1.6

8.0

0.1

RR----

12.8

3.8

2.4

0.8

Nonop

15.2

0.2

0.8

0.6

There were no significant differences in drinking behavior on the water test of Day 2 (Kruskal-Wallis H-test, p>0.1); all rats drank about 12 to 15 ml of water in the 10 rain test. On the first LiCI test on Day 3, animals under unilateral CSD slightly less than the unoperated animals but the difference was not significant (Kruskal-Wallis H-test, p>0.1). On the day following LiCI I, all groups drank between 2 and 7 ml of water which was significantly less than they had drunk on the water test before LiC1 I (the Wilcoxon matched-pairs signed-ranks test, p = 0.005); however, there were no significant differences between the 3 groups (Kruskal-Wallis H-test, p>0.1). On all subsequent water days, the mean intake of all groups was essentially normal, ranging from 10 to 17 ml, with no significant differences between groups. On LiCI II (Day 6), both Group RR- - - - an d Group R R L drank relatively little, indicating that they had each learned the aversion in the one functional hemisphere. Both groups were s i g n i f i c a n t l y different from Group Nonop (Mann-Whitney U-tests, p=0.02 and p<0.001 respectively, but did not differ from each other. Thus the unoperated animals appeared to show greater learned aversion to LiCI than did the animals under unilateral CSD. Perhaps depression of one hemisphere interferes somewhat with the acquisition of a learned aversion. Alternatively, Janebova [12] found a deficit in the taste threshold for quinine in animals under unilateral CSD and such a deficit might account for the difference between the unoperated animals and the animals under CSD here. Whatever the explanation, all the animals drank significantly less on LiCI II than LiC11 (Wilcoxon, p = 0.005), and it is clear that rats under unilateral CSD can discriminate and show a strong aversion to LiC1. On the third LiCI test (Day 9), Group RRL--was depressed on the previously undepressed (trained) side and tested for transfer of the aversion. Group R R - - - w a s not put under CSD. Group Nonop, as in LiCI II, essentially refused to drink. Group R R - - - a g a i n showed a strong aversion but still drank significantly more than Group Nonop (MannWhitney U-test, p=0.008), while Group R R L - - s h o w e d relatively little aversion and drank significantly more than both Group Nonop (Mann-Whitney U-test, p = 0 . 0 0 1 ) a n d Group RR-- -- (Mann-Whitney U-test, p<0.002). Thus, Group RRL-- animals which drank a mean of 12.2 ml on LiC1 1 showed a strong aversion and decreased intake to 1.6 ml on LiCI II when CSD was on the same side. When CSD was switched to the opposite side on LiC1 III, they showed

LEARNED TASTE AVERSION AND CORTICAL SPREADING DEPRESSION a very weak aversion, greatly increasing intake to 8.0 ml but still drinking an amount significantly less (Wilcoxon, p<0.05) than the 12.2 ml of LiCI I. It is unclear why the RR----animals still showed a greater intake of LiC1 than the unoperated animals even when not under CSD on LiC1 III; perhaps the LiC1 aversion is simply not learned as well by animals who have learned it with only one hemisphere functional. On the last LiC1 test, when none of the animals were under CSD, there were no differences between groups (Kruskal-Wallis H-test, p>0.05); all showed a strong aversion. It appears, then, that lithium chloride aversion can be lateralized to the functional hemisphere, although in this experiment the lateralization was not complete and some transfer occurred. Furthermore, it may be that under unilateral CSD, the animal does not learn the aversion quite as well as with both hemispheres functional. EXPERIMENT 2 The second experiment was carried out to replicate and extend the findings of the first. The principle change in procedure of Experiment 2 was to apply KC1 only once to any hemisphere. Because Experiment 1 demonstrated that almost complete aversion occurred after one test with one functional hemisphere, it was possible in Experiment 2 to test for interhemispheric transfer after a one-trial learning experience and thus to avoid repeated CSD tests to the same hemisphere. There are several disadvantages to giving multiple KC1 applications to the same hemisphere. In addition to difficulty in successfully eliciting CSD repeatedly, there is also the possibility of cortical damage [! 1 ], and an increasingly greater possibility that rats can learn because of fluctuations in electrical activity which occur during each spreading depression session [5, 10]. Experiment 2 also tested for the effects of unilateral CSD on the retrieval of an aversion which had been learned with both hemispheres functional. In addition, the effects of unilateral and bilateral CSD on drinking of water were examined. Method

Thirty-nine male Holtzman Sprague-Dawley rats weighing 3 4 0 - 3 5 0 g were deprived of water and pretrained as in Experiment I. Bilateral cannulae were then implanted in 28 animals and 11 remained as unoperated controls. There were 5 groups; Group RL, Group ---L, Group Nonop, Group RR-H20, and Group BB-H20. (B refers to bilateral cortical depression). The animals were given ad lib food and water for 1 day following surgery and were again deprived of water. After 23 hr of water deprivation, a 6 day sequence of daily drinking tests was initiated, with water on Days 1, 2, 4, and 5, and 0.12 M LiCI on Days 3 and 6. The test procedures were the same as in Experiment 1. For Group RL (n=12), 25% KC1 was applied to the right hemisphere o n Day 3 (LiC! I) and to the left hemisphere on Day 6 (LiCI II) to see if transfer of the learned aversion took place. Group ---L ( n = l l ) was given the LiCI I test on Day 3 with both hemispheres functional and the LiCI II test on Day 6 with 25% KC1 on the left hemisphere to determine whether the CSD state affected retrieval of the learned aversion. The unoperated rats (Group Nonop, n=l I ) w e r e simply tested with LiC1 on both Days 3 and 6. Group RR-H20 (n=3) was

8!

included to determine the effects of unilateral CSD on normal water intake in this test situation; CSD was elicited in the right hemisphere for both test Days 3 and 6, but the animals were given only water throughout the experiment. The last group, Group BB-H20 (n=2), was also given only water tests, but under bilateral CSD on both test Days 3 and 6. Results and Discussion

The total amount of liquid each animal drank in each 10 min test was recorded, and the mean amount of LiCI or water consumed in the tests of Days 3 and 6 for each of the 5 groups is given in Table 2. TABLE 2 MEAN AMOUNT (ML) CONSUMED PER 10 MIN TEST Group

N

Day 3 LiCI I

Day 6 LiCI II

RL

12

15.0

13.0

--L

l1

15.2

2.4

Nonop

11

13.4

1.2

RR-H20

3

12.4 (H20)

14.0 (H20)

BB-H=O

2

0 (H=O)

1.0 (H20)

On LiC1 I, there were no significant differences among the three groups drinking LiC1. In contrast, on LiCI II, Group RL drank significantly more than either Group - L or Group Nonop (Mann-Whitney U-tests, p's<0.001). Both Group Nonop and Group - L drank LiC1 on Day 3 with two hemispheres functional and both groups showed clear aversion to LiCI on Day 6, significantly reducing their intake (Wilcoxon p's<0.005) to only 2.4 and 1.2 ml respectively. On the other hand, Group RL, which drank LiC1 on Day 3 with the right hemisphere depressed, failed to transfer the aversion when tested with the left hemisphere depressed on Day 6 and drank 13.0 ml, an amount not significantly different from the Day 3 intake of 15.0 ml (Wilcoxon, p> 0.1). Although both Group Nonop and Group --L showed a clear aversion in LiC1 II, the --L group drank slightly more than the Nonop group (2.4 and 1.2 ml respectively). Since both groups had learned the aversion under the same condition on Day 3, the small difference in performance on Day 6 indicates that the unilateral spreading depression did interfere somewhat with the expression of the learned aversion. This could be due to a CSD effect on retrieval, an effect on change in the stimulus conditions [251, or perhaps a deficit in discriminatory taste ability [ 12]. When animals under unilateral CSD were tested on water consumption (Group RR-H20) , there was no change from the amount consumed on normal days. However, under conditions of bilateral CSD, there was almost complete cessation of drinking behavior as shown by Group BB-H 2 O which drank normally on nondepressed days but drank mean amounts of 0 and 1.0 ml under bilateral depression on Day 3 and 6 respectively. This is in agreement with reports by Schneider [24] and Levitt and Krikstine [14].

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L E H R AND N A C H M A N

Animals under bilateral CSD in the study by Schneider [24] drank a p p r o x i m a t e l y 4 ml of water in 30 min while those in the study by Levitt and Krikstine [ 14] drank only 0.1 ml in a one hr period. GENERAL DISCUSSION The results of Experiments 1 and 2 indicate that animals can learn and retrieve an aversion to LiC1 with one functional hemisphere. More significantly, the results show that the LiC1 aversion can be restricted to a single hemisphere and that when the aversion is learned with one hemisphere depressed there is little or no transfer when the animals are tested with the o t h e r hemisphere depressed. The lateralization of LiC1 aversion under unilateral CSD suggests that the learning of the aversion normally involves cortical participation and that a hemisphere with the cortex depressed does not acquire the one-trial learned aversion. These results differ from those recently reported by Best and Zuckerman [2] who found interhemispheric transfer of a learned aversion and attributed this finding to subcortical mediation. It is often difficult to assess the reasons why different results are obtained in two experiments, particularly when both are testing essentially the same hypothesis with similar procedures. However, there was one major and perhaps several minor differences b e t w e e n the present experiment and that of Best and Z u c k e r m a n [2] which may account for the results. Most i m p o r t a n t was the difference in the strength of the two aversions studied and the consequences of that difference. In the present experiment, only a single trial was necessary to produce a strong learned aversion in which rats decrease their 10 rain intake from about 12 15 ml to about 0 - 1 ml. When that trial was given once with one hemisphere depressed, there was no evidence of transfer when testing occurred with the other hemisphere depressed ( E x p e r i m e n t 2). When two trials were given with one hemisphere depressed, there was a small a m o u n t of transfer to the o t h e r hemisphere ( E x p e r i m e n t 1). The Best and Z u c k e r m a n [2] experiment e m p l o y e d repeated trials, apparently because of their use of apomorphine, a drug which in a n u m b e r of studies has not been as effective as is LiC1 in producing learned aversions [17]. Thus, to produce a learned aversion, they gave f o u r learning trials, which with their design required that KCI be applied unilaterally to the same hemisphere for eight successive days before testing for transfer. It is likely that the repeated application of KC1 may be, in part, responsible for the disparate results. Nadel [18] has pointed out that it is difficult to elicit CSD day after day and Best and Z u c k e r m a n [2] discarded 40% of the animals that began their experiment because depression was not c o m p l e t e or because of edema or a tear in the dura. In their remaining

animals, Best and Z u c k e r m a n [21 reported extensive lesions, which is consistent with the findings of Hamburg, Best, and Cholewiak [11] that there is some noticeable damage to the cortex after one trial of CSD and a distinct cortical lesion in all animals after two or more trials. Even though only animals showing loss of the limb placing response were included in their experiment, it is possible that depression was not as effective over the prolonged training time and that the animals were learning with both hemispheres. It has been found, for example, that behavioral measures of depression, such as limb m o v e m e n t , do not agree well with neural recordings of cortical depression [8]. Thus, with repeated trials of CSD, it would be more likely that there would be times when behavioral measures of depression would not agree with neural measures of depression. A n o t h e r indication that the levels of depression were apparently different in the Best and Z u c k e r m a n [2] experiment and in the present experiment is given by the water intake data of bilaterally depressed rats in the two studies. While we found that bilaterally depressed rats could drink little or no water in 10 min, in agreement with other reports [14, 2 4 ] , Best and Z u c k e r m a n [2] reported their rats drank large a m o u n t s of as m u c h as 1 5 - 2 0 ml in 10 rain, which were the same as normal amounts. The fact that Best and Z u c k e r m a n [2] found extensive drinking even on the first bilateral CSD trial suggests that the level of depression was different in the two studies and that this difference was in addition to any effects of the multiple CSD treatments. The present result showing lateralization of the learned aversion used the taste of LiCI as the CS and a conditioning procedure in which the sickness producing agent (LiC1) as the CS and a conditioning procedure in which the sickness producing agent (LiCI) was ingested. However, the results are not limited to this situation. In other work in our laboratory (Nachman and Koenigshofer, unpublished observations) we have used the conditioning procedure in which rats drink a sucrose solution and are injected with LiC1 to produce the sucrose aversion. This procedure also results in lateralization of the learned aversion when the learning trial is given under unilateral CSD. Thus, it is concluded that single trial learned taste aversions are normally cortically mediated and that these habits are restricted to the functional hemisphere when CSD is applied to the opposite hemisphere. Subcortical mediation of single trial learning was not found in the present study although it is possible that such learning would occur with repeated trials [13] or after cortical damage. Braun, Slick, and Lorden [3] using ablation methods, have also recently shown the involvement of the gustatory neocortex in the mediation of taste aversions.

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LEARNED TASTE AVERSION AND CORTICAL SPREADING DEPRESSION

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