Cortical spreading depression and the problem of motor impairment

Physiology and Behavior. Vol. 3, pp. 849-855. Pergamon Press, 1968. Printed in Great Britain

Cortical Spreading Depression and the Problem of Motor Impairment I. S T E E L E R U S S E L L , H. C. P L O T K I N A N D D. K L E I N M A N

M.R.C. Unit for Research on Neural Mechanisms of Behaviour, University College London (Received 22 June 1968) RUSSELL, I. S., H. C. PLOTKrNAND D. KLmNMAN. Corticalspreading depression and the problem of motor impairment. PHYSIOL.BEHAV.3 (6) 849-855, 1968.--The effect of cortical spreading depression (CSD) on locomotor performance was studied during acquisition and retention of avoidance conditioning in the runway. During acquisition bilateral CSD selectively blocked start latencies, having no significant effect on running times. Where CSD did, on occasion, result in increased running times this was not attributable to a loss of postural reflexes or other motor impairment, but to the presence of competing responses such as freezing or retracing in the alley. It is suggested that CSD produces a learning deficit rather than any motor impairment. Cortical spreading depression responses

Motor impairment

Placing reflexes

IN RECENT YEARS there has been an increased use of cortical spreading depression (CSD) as a technique of reversible and functional ablation. The major concern of this use of CSD has been to investigate the effect of such functional decortication on the learning capacity of the rat. The majority of studies have attributed these learning impairments to the elimination of cortical mechanisms subserving learning [4, 7, 16, 20, 21, 23]. In the case of the learning impairment during unilateral CSD, several reports have specifically noted an absence o f any locomotor deficit following the unilateral elicitation of CSD [5, 18, 19, 20]. In contrast to this position other experiments have attributed the learning deficit following bilateral CSD to a motor impairment [10, 11, 13, 21, 24]. Similar arguments have also been advanced for the hemidecorticate deficit [14]. In considering the possibility of motor impairment resulting from the presence of CSD, Bure~owi [8, 9] observed that there was no interference in unrestrained rats. Normal posture and motor behaviour was maintained due to the presence of intact spinal, bulbar and mesencephalic reflexes. Bure~ov/t did note, however, the loss of tactile placing reflexes during the presence of CSD, an observation that is congruent with the known cortical localization of this reflex, [1, 2, 3]. Whilst a loss of placing reflexes invariably accompanies CSD, it is doubtful if this loss alone would result in a motor impairment that would interfere with performance in such time-dependent tasks as avoidance learning. N o clear statement has been made concerning the nature of the motor impairment that has been suggested as the source of the learning deficit in the CSD animal. The loss of placing reflexes, however, would appear to be a common factor in the observations of experimenters who advocate this position. The concern of the present paper is not to establish the presence or absence of a motor deficit following CSD. Rather the crux of the issue is to examine whether or not the motor

Competing

requirements of any particular learning situation exceed the motor capacities of a rat with either unilateral or bilateral spreading depression. METHODS

Subjects The subjects were 160 male hooded rats, approximately 90 days old at the time of experimentation. All animals were maintained in individual cages with food and water a d libitum, and their body weights were checked daily throughout the experiment.

Apparatus The apparatus has been described elsewhere previously [16, 17]. Briefly it consisted of a runway 6 ft in length, with a grid floor throughout the start box and alley. The shock grid was composed of 0.375 in. brass rods spaced at 0.375 in. intervals. The floor of the goal box was insulated, being covered by a layer of perspex. The CS used was a 78 dB buzzer located above the start box. The shock delivered to the entire grid was a scrambled d.c. current of 1.0 m A from a constant current source. The CS duration prior to shock onset was 5 sec for all groups of animals. Experimental contingencies as well as the recording of latencies and running times were automaticaUy programmed by standard timing and switching circuitry.

Spreading Depression On the day prior to training, the animals were anaesthetised with diethyl ether and a midline incision was made in the scalp which was then reflected from the skull. A 4 mm internal diameter trephine was used to remove a circular piece of bone immediately caudal to the coronal suture and 1-2 m m lateral to either side of the sagittal suture. Care was taken not to 849

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Avoidance conditioning

850

RUSSELL, PLOTKIN AND KL[IINMAN tween sessions. A single daily 40 trial session was given to groups C, D and F. Groups A and C were run with normal cortical function, whereas groups B and D were bilaterally depressed throughout training. Finally, groups E and F were run with normal function for the first two days, and bilaterally depressed on the third day. All animals were allowed a 30 sec exploratory period alter being placed in the start box of the runway. The onset of the first trial was signalled by the simultaneous raising of the door and presentation of the CS. Thereafter 10 sec was given in the start box prior to the onset of every trial. The intertrial interval varied from 75-115 sec.

bruise or rupture the dura. The incision area was then bathed under a dilute solution of chloramphenical and closed by two or three loose sutures. On the following day approximately one hr before testing, the sutures were cut and the incision area exposed. If the condition of the animal was in any way unsatisfactory, e.g. rupture of the dura, infection etc, the rat would be discarded. Twenty min prior to training a pledget soaked in 25 per cent KC1 was applied to the dura overlying the parieto-occipital cortex. After 10 min a fresh application of pledgers was made. Testing was begun a further 10 min later only if the placing reactions had been abolished. Throughout training placing reactions were monitored on each trial and also the condition and position of the pledgets were regularly inspected. Differences between unilaterally and bilaterally depressed animals were solely in terms of whether one or both hemispheres were depressed.

RESULTS

Considering firstly the performance of the individual animals, Fig. 1 shows the trial by trial results of a normal control subject drawn from group C. As can be seen the running times are high on the first two trials due to retracing in the alley. However, on trial 3 the running times drop sharply to approach asymptotic levels throughout the remaining trials of the two sessions. Similarly Fig. 2 gives the performance of a bilaterally depressed animal drawn from group D. As in the case of the normal animal it can be seen that high running times are obtained on the initial trials due

EXPERIMENT I METHOD

The animals consisted of 20 normal and 40 bilaterally depressed rats. The general design of the experiment is given in Table 1. Groups A, B and E were given two 20 trial sessions per day of training, with approximately 3 hr intervening be-

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CORTICAL SPREADING DEPRESSION

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to freezing and retracing in the alley. Isolated regressions were found in the running times, such as can be seen on trials 19, 39 and 44 in Fig. 2. The extent of these isolated deteriorations, which were always due to the presence of freezing or retracing, was such as to badly skew the group mean for these initial sessions. It should be noted, however, that these regressions in running times were relatively isolated cases and were certainly not due to any motor impairment. It is clear from the inspection of Fig. 2 that there is no decorticate motor deficit indicated by the running times. The deficit is in fact almost entirely restricted to the latencies of the start-box exits. This dramatic increase in latencies was not due to the bilateral CSD animal suffering from any difficulty in initiating locomotor activity. On the contrary it resulted from the apparent inability to eliminate responses in the start-box such as circling, persistent entry into comers, freezing or even attempts to burrow through the grid-floor. This failure to exclude such competing GO responses is indicative of a learning impairment rather than a motor deficit.

of the animals support this interpretation, as can be seen in Table 2. The bilateral CSD animals displayed a high incidence of freezing and retracing on the first two sessions, particularly on session I which gave the highest running times. By the last two sessions these competing responses had been almost totally eliminated resulting in the lowered running times. This would suggest that the decorticate rat is capable of learning, although it is difficult to make a firm statement in the absence of sensitization control groups. It is possible that this loss of competing behaviour was not due to learning but was instead a consequence of the prolonged exposure to shock.

TABLE 2 TOTALNUMBEROF COMPETING-RESPONSES(FREEZINGAND RETRACING) IN EACHSESSIONFOR Not~tAt,S AND BILATERALLYDEPRESSED (CSD) ANIMALS Day 1 Sessions

TABLE 1 AVERAGE SESSION RUNNING TIMES (IN 0.001 rain) FOR NORMALS AND BILATERALLYDEPRESSED (CSD) ANIMALS Day 1 Sessions Groups A B

I

Day 2 II

III

IV

CSD 51

CSD 22

CSD 28

E

Normal 25

Normal 21

Normal 23

Normal 23

Normal 22

Normal 36

F

CSD 25

CSD 39

CSD 34

The individual results illustrated in Figs. 1 and 2 were not in any way atypical as can be seen in Table 1, which gives the mean session running times for all groups. A comparison of group A and group B shows that whilst the bilateral CSD running times are greater than those of normals for the first two sessions of day 1, nevertheless the bilateral CSD running times are well within the normal range for sessions III and IV. If the poor performance on the first two sessions were attributable to a motor impairment due to CSD, then it would seem that the marked improvement on the last two sessions is suggestive of some sort of compensation. This interpretation is, however, unlikely when the results of Fig. 2 are considered. It would appear that the motor requirements of the task are well within the motor capacity of the bilateral CSD animal. The initial poor performance is due to the rats having to acquire the response of smoothly traversing the alley from start-box to goal-box by eliminating such competing behaviours as freezing and retracing. Qualitative observations

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Normal 13

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As the running times for both normal and CSD animals are not normally distributed, it is possible that the use of mean scores in Table 1 tends to exaggerate the differences between groups. A more complete picture of the running time profiles of the decorticate animals in group B is given in Fig. 3, where the frequency of occurrence of any given running time is expressed as percentages of the total trials. On the first session two prominent peaks are present in the bilateral CSD running time profile. The first peak, or the mode, lies in the third interval, showing that almost 50 per cent of the responses were being performed with running times of approximately 1.5 sec. The other peak occurs in the last interval, where all running times greater than 5 sec axe pooled. These response times are comprised entirely of the freezing and retracing responses given in Table 3. Neither of these two response peaks is indicative of a motor deficit, which would be expected to produce a cluster lying midway in between the two extremes. By the second session the peak coinciding with competing responses has disappeared, hence the decrease in mean running times (Table 1) which continues over the remaining sessions. As the sessions progress so the modal running time decreases to approximately 1.0 sec. The running time profiles of bilateral animals can be contrasted to those of group E in Fig. 3. The first four sessions when the animals are normal can be used therefore as a direct

852

RUSSELL, PLOTKtN AND KL[~INMAN

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comparison with the bilateral CSD animals in group B. (The results given in Tables 1 and 2 indicated the very close correspondence between normals in groups A and E). On the first session the normals in group E show a clustering of running

times at the short intervals. As the sessions progress, however, there is a marked tendency for these fast runs to diminish as the frequency of slightly more prolonged running times increases. This trend probably reflects a stabilising of avoidance

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CORTICAL SPREADING DEPRESSION behaviour where the running times become somewhat more relaxed in nature or slightly longer. The result of these opposed changes in normal and bilateral CSD modal responses is that by the third and fourth sessions the bilateral CSD animal's running times are marginally faster than those of the normals. Considering the average running times in Table 1, the bilateral CSD animals had appeared to be slower than normals. However, it is clear from Fig. 3 that this use of the average for such skewed distributions would be inappropriate. Any minor changes in the frequency of extreme values would introduce appreciable distortions in the mean value. It is of additional interest to note in Fig. 3 that when the normals in group E were rendered functionally decorticate the running times for the fifth and sixth sessions conformed to the practiced profile of the bilateral CSD animals in group B. These results are in no way supportive of the notion of a bilateral CSD motor deficit. The effect of giving a single daily 40 trial session is neglible for normals but very marked for bilateral CSD rats. As can be seen in Table 1 the bilateral running times of group D are considerably reduced on the first session when compared to those of group B. Both groups D and B are indistinguishable from normal controls on the second day. The single 40 trial session has resulted in a shorter mean running time due to the lower incidence of competing responses. As can be seen from Table 2 the chief advantage of a single long session over two short sessions is that there is a recurrence of competing responses at the start of the second short session. The running time profiles are given in Fig. 4 for groups D and F. Here it can be seen that the proportion of slow running times is considerably reduced for the bilateral CSD animals in group D by comparison to the first session of the bilateral CSD rats in group B (Fig. 3). Further on the second session, the bilateral CSD animals show a pronounced peaking of running times at the 1.0 sec interval. When compared to the normal profiles of group F, the modal running time is again less than that of normals. Finally when group F on the third session is run functionally decorticate, the profile in Fig. 4 reverts to that of a practiced bilateral CSD animal. The results of groups E and F, as seen in Figs. 3 and 4, and to a lesser extent in Table 1, are very striking. These groups show that prior training with normal cortical function facilitate performance within the alley when the animal is subsequently functionally decorticated. If bilateral CSD were to result in impaired motor control then the running times should increase with the presence of CSD, irrespective of prior training in the normal state. The present findings clearly do not support this viewpoint. Furthermore, the low running times in the CSD phases of groups E and F argues against the possibility of any compensation occurring with repeated CSD treatments.

EXPERIMENT H From the results of the previous experiment it is apparent that bilateral CSD did nor result in a motor deficit sufficient to reduce performance and contribute to the decorticate learning defect. However, a considerable effect was seen on the performance of the bilateral CSD animals during the initial session when the trials were massed during training. A second experiment was undertaken to attempt to systematically evaluate this finding in terms of the effect of different intertrial intervals on runway performance during CSD. For the sake of completeness the performance of both unilateral and bilateral CSD animals was compared to that of normals.

853

METHODS

The animals used were 100 male hooded rats maintained in the same fashion to those in Experiment 1. The apparatus and spreading depression treatment were the same as previously described. The general design and procedure of the experiment has been reported in detail elsewhere [16]. Briefly, the animals were divided into three main groups of normal controls, unilaterally depressed and bilaterally depressed animals. These in turn were subdivided into 4 subgroups corresponding to the intertrial intervals (ITI) o f 15, 30, 60 and 240 sec. Each subgroup consisted of 8 animals, with the exception of the bilateral CSD 15 sec I T / g r o u p which contained 12 animals. On each of the two days of training the animals were given a single block of 20 trials. In all other respects the experimental procedure was identical to Experiment I.

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RESULTS

The mean session running times are given for all groups in Fig. 5. Considering the 30, 60 and 240 sec IT[ groups it is clear that on day 1 a graded effect on running times is suggested. That is the controls are superior to unilaterals, which in turn are superior to bilaterals. An examination of the incidence of such competing responses as freezing and retracing reveals a similar graded relationship as can be seen from Table 3. Such competing tendencies, which cannot be equated with any form of motor impairment, suggest that the day 1 differences in running times for CSD animals must be due to some central deficit in learning. By day 2, differences

854

RUSSELL, PLOTK1N AND KLEINMAN

in amount of competing behaviour had been considerably reduced, and consequently the running times for all groups were similar. This observation was supported by a three factor analysis of variance, where the three-way interaction was significant beyond the 5 per cent level (F -- 2.8408, d f - : 6).

TABLE 3 TOTAL NUMBER OF COMPETING RESPONSES (FREEZING AND RETRACING) IN EACH SESSION FOR NORMAL, HEMIDECORTICATE AND DECORTICATE ANIMALS AT DIFFERENT INTERTRIAL INTERVALS (ITIs)

Day 1 Freeze Retrace

Day 2 Freeze Retrace

ITI

Cortex

30 sec

Normal Hemi Decort

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Normal Hemi Decort

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0 2 2

0 3 1

240 sec

Normal Hemi Decort

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1 5 4

0 1 2

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Considering the performance of the 15 sec ITI groups in Fig. 5 it is seen that the performance of the bilateral animals is superior to that of unilateral animals and well within the normal range. This finding is congruent with the latencies yielded by this group [16], and arises from the short ITI producing in the decorticate a stereotyped short latency escape response within very few trials after the onset of training. The salient point is that the bilateral CSD animal de novo is capable of as rapid and coordinated motor behaviour as the normal controls under these conditions. On day 2 the mean running times for all three preparations were similar. Indeed, the 15 sec ITI bilateral CSD group gave the lowest average running time in the entire experiment. This finding is in close agreement with the results of the second day performance of the bilateral massed practice group in Experiment 1. DISCUSSION

The present results clearly showed that the CSD animal manifests no motor deficit thatches sutticient to detract from performance in the present test situation. Where running time were found to be increased this was invariably due to the presence of competing responses. The motor requirements of the task were seen to be well within the motor capacity of either the unilateral or bilateral CSD animals. The presence of an acquisition deficit for CSD animals, presumably due to a loss of cortical function, also cannot be attributed to any motor impairment. A reconsideration of the previous assertions of a motor deficit in the CSD animal becomes necessary in the light of the present findings. The earliest suggestion of a motor impairment in the bilaterally depressed rat came from Tapp [22], who predicated his case upon the results of a test in which animals were required to maintain their equilibrium when placed on a slowly rotating rod. A significant relationship was found between failure on this test and a lack of retention of a shuttle avoidance response under bilateral CSD. On this basis Tapp con-

cluded that the loss in avoidance retention was related to a "general motor impairment". This conclusion is purely conjectural as the stick test only provides a measure of the integrity of complex cortical postural reflexes [6]. There is no reason to believe that these reflexes constitute a critical "link'" in the locomotor pattern involved in shuttle-box avoidance. A more reasonable interpretation would be that the loss of cortical postural reflexes due to CSD indicates merely a loss of cortical function. The retention loss following functional decortication would then be indicative of the cortical role in learning and memory. The present results make it clear that a retention deficit could not be due to any motor impairment. Mogenson [14] observed that CSD animals tended to be lethargic, and further noted that if a limb contralateral to the depressed hemisphere in unilateral CSD rats dropped between the bars of the grid floor then it was not placed. On this basis the avoidance deficit of depressed animals was attributed to a motor impairment. The observations of lethargy would seem to be more suggestive of a motivational effect rather than any motor impairment. Further the failure to retrieve a fallen limb is indicative of a loss of placing reflexes, which need be in no way detremental to locomotion. We have frequently observed a failure to place a fallen limb with CSD rats in the runway. With only very rare exceptions, it has not been found to detract from the performance of the response since the limb can be retrieved promptly by general body movements following the presentation of the CS or shock. The incidence of such failures to retrieve a limb has been found to occur about once every 400-500 trials. This has been most commonly observed to occur when the animal has failed to avoid and has wrenched itself around to the shock. In any event the frequency of such occurrences is too low to be in any way contributory to any decorticate deficit. Winocur [24] claimed to provide support for Tapp when he reported a greater acquisition impairment in bilaterally depressed rats to avoid via a small door in a Yerkes-Thompson box, as compared to avoidance via a larger door. This difference produced by task difficulty need not however be due to the BSD animal's limited motor capacity. Koranyi, Endroczi and Lissak [12] have reported coordinated escape responses in bilateral CSD rats which were required to jump onto a small platform that was 15 cm above floor level. As this height is more than twice that of the small door employed by Winocur, it is unlikely that the bilateral impairment was due to motor impairment. It is more likely that his results reflect a central learning deficit, of the kind so clearly manifested in the latencies shown in Fig. 2. Freedman and Lash [11] found that the latencies of bilateral CSD rats increased over trials during avoidance training and attributed this acquisition debility to a motor deficit produced by CSD. As their report contained no observations on running times it is likely that these results reflect the same sort of learning impairment that has been seen in Fig. 2. Furthermore the fact that the latencies deteriorated with practice is supportive to this interpretation. It is difficult to conceive of a motor deficit that increases in severity with practice without the presence of cumulative trauma, Finally, Meyers and Stern [13] observed a reduction in general activity following bilateral CSD which they attributed to a motor impairment. No distinction was made between an activity reduction due to a motor deficit, or because of a decreased level of reactivity on the first session of bilateral CSD. It is interesting to note in this regard that Nadel [15] tested bilaterally depressed rats for four consecutive sessions and observed an increase in activity as the sessions continued.

CORTICAL SPREADING DEPRESSION

855

It is clear that the problem of whether the CSD rat suffers from some sort of impaired motor function is both complex and to some extent confused. Terminological confusion and vagueness has occurred by equating lethargy reduced activity and prolonged latencies with motor impairment. This is not only false but also misleading. Although certain corticallybased postural reflexes are lost during CSD, the resulting motor impairment will not necessarily interfere with the

animal's performance in a learning situation. To demonstrate that a motor deficit impairs learning it is essential to show a causal relationship between them. The present results show that in spite of a considerable learning impairment the motor ability of the animal is not critically affected. It would appear that the loss of cortical postural reflexes per se is irrelevant to the motor requirements of the learning tasks in these experiments.

REFERENCES 1. Bard, P. and C. M. Brooke. Localised cortical control of some postural reactions in the cat and rat together with evidence that small cortical remnants may function normally. Res. publ. Ass. herr. ment. Dis. 13: 107-157, 1934. 2. Braun, J. J. The neocortex and visual placing in rats. Brain Res. 1: 381-394, 1966. 3. Brooks, C. M. Studies on the cerebral cortex. I1. Localized representation of hopping and placing reactions in the rat. Am. J. Physiol. 105: 162-171, 1933. 4. Bureg, J. Reversible decortication and Behavior. In: The C.N.S. and Behavior, edited by M. A. B. Brazier. J. Macy Jr. Foundation, New York. (1959), pp. 207-248. 5. Buret, J. and O. Buregovfi. The use of Leap's spreading cortical depression in the study of interbemispheric transfer of memory traces. J. comp. physiol. Psychol. 53: 558-563, 1960. 6. Bureg, J. and O. Bure~ova. The use of Leap's spreading depression in research on conditioned reflexes. Electroenceph. clin. Neurophysiol. Supp. 13: 359-376, 1960. 7. Bureg, J., O. Buregov/t and A. Zaharov/~. Conditioned reflexes and Leap's spreading cortical depression. J. comp. physiol. Psychol. 51: 263-268, 1958. 8. Bure~lovA, O. The effect of non-conditioned and natural conditioned reflexes on the course of spreading EEG depression. Physiol. Bohem. 5: 350-358, 1956. 9. Bure~vA, O. Influencing water metabolism by spreading EEG depression. Physiol. Bohem. 6: 12-20, 1957. 10. Delprato, D. J. A note of the effect of cortical spreading depression on open field behavior. Psychol. Rep. 17: 714, 1965. 11. Freedman, N. L. and L. Nash. Conditioned avoidance decrement under spreading depression. Psychonom. Sci. 5:411-412, 1966. 12. Koranyi, L., E. Endroczi and K. Lissak. Disinhibition of extinguished conditioned reflex under spreading depression. Acta physiol. Akad. Sci. Hung. 27: 353-357, 1965.

13. Meyers, B. and W. C. Stern. Effect of bilateral spreading depression and scopalamine on motor activity in rats. Psychol. Rep. 18: 267-270, 1966. 14. Mogenson, G. J. Effects of spreading cortical depression on avoidance responses conditioned to peripheral or central stimulation. Electroenceph. clin. Neurophysiol. 18: 663-669, 1965. 15. Nadel, L. Cortical spreading depression and habituation. Psychonom. Sci. 5: 119-120, 1966. 16. Plotkin, H.C. Role of the neocortex in acquisition of avoidance conditioning. Nature 213: 1053-1054, 1967. 17. Ross, R. B. and I. S. Russell. Lateralization and one-trial interhemispheric transfer of avoidance conditioning. Nature 204: 909-910, 1964. 18. Rudiger, W. and Bureg, J. Cortical spreading depression and tegmental and hypothalamic threshold stimulation producing locomotor flight. Physiol. Bohem. 11: 399--403, 1962. 19. Rudiger, W. Interfering subcortical stimulation and cortical spreading depression. Physiol. Bohem. 11: 392-398, 1962. 20. Russell, I. S. The differential role of the cerebral cortex in classical and instrumental conditioning. In: Biological and physiological problems of psychology. XVIII Int. Cong. Psychol., Moscow, p. 115. 21. Russell, I. S. Animal learning and memory. In: Aspects of learning and memory, edited by D. Richter. London, Heineman Medical Press, pp. 121-171, 1966. 22. Tapp, J. T. Reversible cortical depression and avoidance behavior in the rat. J. comp. physiol. Psychol. 55: 306-308, 1962. 23. Travis, R. P. and D. L. Sparks. The influence of unilateral and bilateral spreading depression during learning upon subsequent learning. J. comp. physiol. Psychol. 56: 56-59. 1963. 24. Winocur, G. Bilateral spreading depression and avoidance learning in rats. Psychonom. Sci. 3: 107-108, 1965.