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The effect of a twenty-four hour intertrial interval on the acquisition of spatial discrimination by hippocampally damaged rats
Physiology and Behavior, Vol. 8, pp. 457-462. Brain Research Publications Inc., 1972. Printed in Great Britain
The Effect of a Twenty-four Hour Intertrial Interval on The Acquisition of Spatial Discrimination by Hippocampally Damaged Rats I L A R R Y W. M E A N S L M I C H A E L
L. W O O D R U F F s A N D R O B E R T L, I S A A C S O N
Department o f Psychology, University of Florida, Gainesville, Florida, 32601, U.S.A. (Received 18 A u g u s t 1971) MEANS, L. W., M. L. WOODRUFFAND R. L. ISAACSON.The effect of a twenty-four hour intertrial interval on the acquisition of a spatial discrimination by hippocampally damaged rats. PHYSIOL.BEHAV. g (3) 457--462, 1972.--Rats with either
aspiration- or penicillin-induced (irritative) lesions of the hippocampus or overlying neocortex and normal rats were trained on a spatial discrimination in a T-maze after being tested for spontaneous alternation. Animals were given only one trial per day (24 hr ITI). Only the animals with aspiration lesions of the hippocampus were impaired in acquiring the spatial discrimination. Animals with either hippocampal or neocortical lesions alternated at reduced rates. Hippocampal lesions Neocortical lesions Twenty-four hour intertrial interval
Penicillin-induced epileptogenic foci
THE PRESENT study was undertaken to determine the effects of aspirative and irritative lesions of the hippocampus on acquisition of an appetitive task. The experiments of Olton [16] and Schmaltz [18] have suggested that the discrepancies observed between some of the behavioral effects produced in certain epileptic patients by temporal lobe destruction [13, 14] and those produced in animals by experimental destruction of the hippocampus [1] might be due to the epileptic condition of the brains of the former. In their studies with animals with penicillin injected into hippocampus, Olton and Schmaltz found electrographic signs of epileptiform activity and also demonstrated impairments in the acquisition of a two-way active avoidance problem. Animals with radical bilateral hippocampal destruction, on the other hand, are known to acquire the two-way active avoidance task more rapidly than normal animals [5]. The results of these studies led us to believe that it was important to compare the effects of the two types of lesions in other types of tasks. The task selected for this experiment, a spatial discrimination, is one in which hippocampally ablated animals perform in the same manner as normal animals [7, 15], provided the intertrial interval (ITI) does not exceed 20--30 min [9, 10]. Since the intertrial interval (ITI) is influential in determining the behavior of animals with hippocampal destruction, we attempted to maximize this aspect of the training with a
Spatial discrimination
24 hr 1TI, i.e., giving one trial per day. In addition, since it has recently been shown that rats with hippocampal lesions use fewer strategies of hypotheses than normal rats in acquiring brightness discrimination [8, 19], it was decided to examine carefully the choice behavior of the animals during acquisition. Finally, we wanted to determine the effect of the penicillin-induced irritative lesions on spontaneous alternation. Accordingly, all animals were tested for spontaneous alternation prior to spatial discrimination learning. METHOD
Animals Fifty Long-Evans male rats, weighing between 304 and 413 g at the time of surgery, were used. The animals were housed individually under conditions of continuous light.
Surgery The animals were divided into 5 groups of 10 animals each. The hippocampal aspiration group (HA) received bilateral aspiration lesions of the hippocampus, as well as of the overlying neocortex, while the animals in the cortical aspiration group (CA) received ablations of only the neocortex overlying the hippocampus. The aspiration procedure employed has been described previously [5]. The hippocampal
1Supported in part by NIMH Grant 16384-03 to R. L. Isaacson and USPHS training grant MH-10320 to Center for Neurobiological Sciences, University of Florida Medical Center. The authors wish to thank Mrs. Pauletta Sanders for her excellent preparation of histological materials. SNow at Department of Psychology, East Carolina University, Greenville, North Carolina, 27834. 3Requests for reprints should be sent to Michael Woodruff, Department of Psychology, University of Florida, Gainesville, Florida, 32601. 457
458 penicillin group (HP) received bilateral implants of penicillin (Parke, Davis and Co.; Type S-R) in the hippocampus and the cortical penicillin group (CP) received similar bilateral penicillin implants in the overlying cortex. The penicillin implant procedure has been described by Olton [16]. The stereotaxic coordinates, used for both implant groups, were anterior 1.8 and lateral 5.1 [17]. The penicillin was placed 4.9 and 0.8 mm below the surface of the cortex in the HP and CP groups, respectively. All surgery was performed under asceptic conditions using sodium pentobarbital anesthesia (50 mg/kg). The control animals (group N) received no lesions.
Apparatus All behavioral testing was conducted in a wood T-maze painted a fiat gray. It consisted of a 8 × 4 in startbox, a 4 × 10 in runway, and two 4 × 18 in arms. All sections were 6 in deep and covered with k in mesh hardware cloth. Guillotine doors separated the startbox from the runway and the arms from the choicepoint. The tops of both choicearms of the maze were covered with cardboard.
MEANS, WOODRUFF AND ISAACSON seizure-like discharges which resemble those found after the application of penicillin to neural tissue [6]. The recording sessions began 20 min following the lowering of the electrodes. Olton [16] and Schmaltz [18] have observed injury spikes as a result of placing the electrodes in the hippocampus, but these spikes continue only a few minutes and are easily differentiated from the spike activity produced by penicillin.
Histology After completion of behavioral and electrophysiological testing, all operated animals were sacrificed and intracardially perfused with 0.9 per cent saline and I0 per cent formalin. The brains were removed, embedded in celloidin, and sectioned at 30 ~t. Every tenth section of the aspiration animals' brains and every second section of the brains of the penicillinimplanted animals was stained with thionin and examined for the locus of the drug implant or the extent of the lesion.
RESULTS
Histology Procedure Preliminary handling and training was begun 11 days preoperatively. On the first 3 days of this period all animals were handled in groups of five to seven on a table for a period of 30 min. The deprivation schedule was initiated on Day 3. Except for the day prior to surgery and for 14 days postoperatively, all animals were maintained at 8 5 ~ of their ad lib body weight throughout the experiment. They were weighed and fed immediately following their daily handling or test session. On Days 5-7 of the preoperative period each rat was handled individually and exposed to Noyes 0.045 g food pellets placed in a small dish on top of the handling table. On days eight and nine preoperatively, all rats were allowed to individually explore the T-maze for 5 rain, or until the 4 Noyes pellets placed in the food cup of each arm were consumed, whichever occurred first. Spontaneous alternation testing was conducted on the 14th to 16th postoperative days. All rats were run each day until they completed six trials or until they failed to make a choice within 5 min of being placed in the maze. Upon making a choice, the animals was retained in the goal box for 20 sec and then given another trial. Food pellets were not present in the maze during spontaneous alternation training. On postoperative Days 17 and 18 four 0.045 g Noyes food pellets were placed in both choice-arms, and the rats were allowed to individually explore the maze for 5 rain, or until they consumed all the pellets, whichever came first. Spatial discrimination training was initiated on postoperative Day 19. Each rat was given one non-correction trial each day. All rats were trained to their nonpreferred side, namely, that side which was chosen least frequently on the first trial of each of the three alternation days. Four 0.045 g Noyes pellets constituted the reinforcement for a correct choice. All rats were run until they made a correct response on 10 consecutive trials. Upon completion of behavioral testing bipolar electroencephalographic recordings were made from the sites at which penicillin had been placed in seven of the HP and five of the CP animals. All recordings were done with the animals placed in a stereotaxic instrument while anesthetized with a 1 5 ~ urethane solution (1.75 g/kg, IP). Urethane, in the amounts used in this experiment, does not in itself produce
Examination of the brain sections revealed that animals in the hippocampal lesion group had undergone almost total removal of the dorsal and lateral hippocampus; various amounts of the ventral hippocampus were spared in different animals. The neocortex and capsule fibers overlying the hippocampus were destroyed bilaterally. The posterior edge of the lesion typically extended into the entorhinal cortex. Slight unilateral thalamic damage was found in three of the ten subjects. In five animals of this group bilateral degeneration of portions of the dorsal lateral geniculate nucleus of thalamus, as well as the lateral and posterior thalamic nuclei was observed. No behavioral effects could be correlated with the position, or presence, of thalamic degeneration. No thalamic degeneration was observed in the cortically ablated animals. The degeneration could possibly be due to involvement of the optic radiations in the course of removing the hippocampus. Examination of the lesions produced in the cortical group revealed that an amount of neocortex comparable to that removed in the hippocampal ablations had been removed. One animal in this group was an exception in that unilateral hippocampal destruction was found. This animal and one other animal from this group that died during the course of the experiment were eliminated from the data analysis. In the penicillin-implanted animals microscopic examination revealed that the penicillin produced small lesions of about 1 mm in dia. Reconstructions of the maximum and minimum extent of the aspirative lesions and the placements of the penicillin are presented in Fig. 1. Figure 2 shows photomicrographs of representative neocortical and hippocampal penicillin implants.
Eleetroencephalographic Results Abnormal electrical activity was recorded from the site of drug application in five of the seven animals from which recordings were made with hippocampal placements and from three of the five animals with neocortical placements from which recordings were made. The abnormal activity, recorded at the end of training, differed from that observed immediately following penicillin implant, when sharp spikelike activity appeared against a depressed background. Figure 3 shows the representative E E G from a pilot animal
FIG. 2. Photomicrographs of representative cortical (upper) and hippocampal (lower) penicillin lesions.
(facing page 458)
HIPPOCAMPAL LESIONS AND INTERTRIAL INTERVAL
459
FIG. 1. Minimum (darkened area) and maximum (cross-hatched) lesions extents for group CA (upper left two rows) and group HA (lower left two rows). Penicillin placements for group CP (upper right two rows) and group HP (lower right two rows).
immediately following placement of penicillin in the hippocampus (bottom 2 traces). The records from one hippocampally-implanted animal at the end of training showed no sharp waves in the EEG. Two of the hippocampallyimplanted animals and one cortically-implanted animal that did show spike-activity are also included in this figure. There was, however, no correlation between the presence or absence of epileptiform activity and the performance of an animal on the behavioral tests utilized in this study.
Behavioral Results The mean alternation rate of five groups is shown in Table 1. In agreement with the findings of Douglas and Isaacson [2] and Means, Leander, and Isaacson [11], the H A group made significantly fewer spontaneous alternations than did the N, CP, or HP groups (one-way analysis of variance F - - 7.68, dr= 4,42, p < 0.01). However, an a posteriori Newman-Keuls analysis [23] indicated that both the CA and the H A group alternated at a significantly lower rate than the other three groups (p < 0.01 in all cases). F o r the analysis of the spatial discrimination task, the number of trials for each rat to reach successive criteria of eight correct responses in 10 trials, nine correct responses in 10 trials, and 10 correct responses in 10 trials were examined (Table 1). A one-way analysis o f variance indicated a significant difference on both the 8/10 and 9/10 criterion measures (F ~ 6.76, d f = 4,43, p < 0.01 ; F ---- 3.49, d f ~ 4,43, p < 0.05), but no differences were found using the 10/10 criterion. A Newman-Keuls analysis was performed on the data of the two significant criteria and the H A group was found to be significantly different (p < 0.01) from the other four groups, which did not differ from each other. Further analysis of the data indicated that the H A subjects made significantly more errors before reaching the terminal criterion of 10 correct responses out of 10 trials than did the CA, N, HP, or CP groups (F ~- 2.96, d f = 4,43, p < 0.05). Figure 4 presents an analysis of the frequency with which sequences of consecutive responses were made to the rewarded (defined as originally non-preferred) and non-rewarded (defined as originally preferred) sides of the T-maze. The ten consecutive rewarded responses which comprise the
terminal criterion are omitted from this analysis. Except the group suffering hippocampal lesions, all groups exhibited very similar patterns of response sequences. The number of sequences of one response to either the rewarded or nonrewarded side of the maze were essentially equal for the hippocampally lesioned animals, while the other groups made significantly more single responses to the non-rewarded (preferred) side. Chains of responses of more than two in a row were made most frequently to the rewarded arm of the maze by all groups except the hippocampally ablated group, which exhibited more sequences of responses to the rewarded non-preferred arm only for sequences of three or more consecutive responses. It should also be noted that the hippocampally lesioned animals differed from the other groups in that seven of the ten animals in this group exhibited long chains of responses to the preferred arm of the maze. However, the animals with hippocampal destruction which did not exhibit long strings of incorrect responses learned the same problem as quickly and exhibited the same response patterns as did animals in the other groups of subjects. Examinations of the behavioral results (Table 1) indicates that neither the HP or CP group differed from group N, the normal control group, on either spontaneous alternation or spatial discrimination. Analysis of variance and subsequent Newman-Keuls confirm these observations. Groups HP, CP, and N were not found to differ on any of the behavioral tests. DISCUSSION
Animals in group H A took significantly more trials to attain the 8/10 and 9/10 criteria and made significantly more errors before reaching the terminal criterion of 10/I0 than all other groups tested, clearly indicating that hippocampal ablation produced a deficit in the acquisition of this task. Other studies examining the effects of hippocampal lesions on spatial discrimination have failed to observe a deficit with short intertrial intervals [7, 9, 15]. However, Means and Douglas [10] observed a deficit in hippocampally ablated rats in a situation where most of the animals were trained to their non-preferred side. Kimble and Kimble [7] also trained their animals to the non-preferred side and failed to
460
MEANS, WOODRUFF AND ISAACSON TABLE 1 MEAN PER CENT SPONTANEOUS ALTERNATION AND MEAN NUMBER OF TRIALS TO SUCCESSIVE CRITERIA ON SPATIAL DISCRIMINATION
Task
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FIG. 3. Top 8 traces present representative electrical activity from site of penicillin implant recorded after completion of behavioral testing. Top 2 traces: right (R) and left (L) cortex of animal implanted cortically with penicillin. Middle 6 traces: right and left hippocampus of animal implanted with penicillin in the hippocampus. H34 and H36 exhibit abnormal sharp waves while H37 does not. Bottom 2 traces: right and left hippocampus e r a control rat immediately following implantation of penicillin.
observe a deficit on the original acquisition of a spatial discrimination in a Y-maze. These latter authors determined the animals' non-preferred arm of the maze in quite a different manner from that used in the present study. They determined the non-preferred arm on the basis of the responses made on the first five trials given on the first training day. Rewards were present in both arms of the maze on these trials. Formal training was begun on trial six. Therefore, we cannot exclude the possibility that the training to the non-preferred side, measured by the first response made each of three days during the spontaneous alternation tests, was a factor in the present experiment. The situation is further confounded by the fact that Means and Douglas [10] gave their four preacquisition test trials during which the first response made by the animal
Spontaneous Alternation Spatial Discrimination
Measure
~'d, 8/10 9/10 10/10
HA
CA
Group N
HP
CP
51 39.8 43.3 48.2
48 15.6 17.3 26.4
81 15.5 21.I 40.5
78 17.5 28.2 39.6
84 12.6 21.0 36.5
was reinforced. Kimble and Kimble [7] reinforced responses to both arms of the maze when determining the animals' preferences. The present study did not differentially reinforce the animals prior to acquisition training. The present study differs from previous studies in that it employed a much longer ITI, 24 hr. The longest ITI of which we are aware that has been employed with hippocampally ablated rats is 30 min [10]. The second possible explanation of the present results is that the deficit stems from the long intertrial interval used. Such an explanation would be consonant with the results reported from the study of an epileptic patient who has had the mesial portions of the temporal lobes resected [13]. This patient can remember a series of digits for several minutes if not disrupted. If a disrupting stimulus, such as a comment by the experimenter occurs, causing a shift in attention, the patient forgets the digits as well as the retention task itself. Possibly the longer ITI allows more opportunity for disrupting stimuli to produce attention shifts and subsequent memory deficits. Consistent with this hypothesis, hippocampally ablated rats have been shown to be deficient on a single alternation task when ITIs longer than 20 sec are employed [21, 22] or when another problem is introduced during the ITI [12]. Also, Hirsch [4] has recently suggested employing a memory hypothesis to explain reduced variability by hippocampectomized rats in a four choice situation. One difficulty with a simple hypothesis based upon an enhanced rate of memory loss over time is the fact that the animals with aspirative lesions of the hippocampus do not choose randomly between the arms of the maze. If each response was made without regard to the previous response(s), i.e., with no memory of the past at all, the long sequences of ten and more responses, which mark the performances of most animals with hippocampal damage, made to the preferred side of the maze would be difficult to explain (see Fig. 4). Moreover, it is obvious that animals with hippocampal lesions do retain information from one day to the next. Animals trained in a spatial discrimination task with shorter ITIs which acquire the problem carry over the information from day to day. Therefore, if the effect of the hippocampal lesion is to disrupt retention over extended time-periods then this deficit is specific to a certain kind of information related to the reward or non-reward of a response and not about the solution to the problem. The analysis of the frequency of correct and incorrect response sequences was undertaken to reveal any differences between the impaired performance of subjects with hippocampal lesions and the performance of animals in other
HIPPOCAMPAL LESIONS AND INTERTRIAL INTERVAL
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FIG. 4. The average frequency of sequential responses to either the non-reward (solid bar) or rewarded (striped bar) arm of the maze is indicated on the ordinate of each graph. The abcissa indicates the number of responses included in each sequence.
groups. We believed that comparing sequences to the preferred and non-preferred sides would enable us to evaluate the possibility that animals with hippocampal lesions had a side, position, or response preference. All of these were confounded in the present experiment and such preferences might have been more difficult to overcome for hippocampally ablated animals than for other animals. We reasoned that this preference could be expressed as an altered probability of response to the two arms of the maze in the presolution period. Widely discrepant response probabilities should be revealed in widely different frequencies of occurrence of strings of consecutive responses of different length, assuming independence of responses. For example, if the probability response to the right arm was 0.8 then the probability of making two consecutive responses to the right arm would be 0.64, while the probability of two consecutive responses to the left arm would be only 0.04. If the probability of response to the two arms was equal, the probability of two consecutive responses to either arm would be 0.25. There was some reason to believe that animals with hippocampal lesions might have a different or
stronger side preference since all but one animal in this group had a preference for the right arm of the maze while animals in other groups distributed their preferences more evenly between the two arms. However, no evidence for a differential tendency to respond to the preferred arm was found for these animals, and in fact their single responses were more equally divided among the preferred and nonpreferred arms of the maze than by animals in other groups. The fact that all animals made more consecutive responses to the non-preferred (rewarded) arm than the preferred (non-reward) arm in the pre-solution period suggests that reward influences the animals' behavior before the beginning of the criterion block of trials and that this compensates for any preferential tendency to respond to the preferred side. The group of animals with hippocampal lesions differed from the other groups in that animals in this group were more 5kely to make long series of incorrect responses, always to the preferred side. There were animals in this group, however, that were unimpaired in acquiring the problem and produced behavior similar to that of the other groups. It becomes apparent, then, that the behavioral impairment
462
MEANS, WOODRUFF AND 1SAACSON
produced by the lesion was made manifest by a greater likelihood that individual animals would make one or more long sequences of incorrect responses. The effect was all-ornone. Either the animal made a long sequence of errors (10 or more) or it did not. The frequency of response sequences of intermittent length is no different than that found in other groups of animals. This observation of all-or-none behavioral deficits among a group of animals suffering from hippocampal lesions has recently been emphasized by Thomas [20]. The finding that hippocampally ablated rats lack the normal tendency of a rat to spontaneously alternate has often been reported in the literature, but the results of the present study indicate that cortically lesioned animals may also alternate significantly less than normals. It should be noted, however, that other studies employing similar cortical lesions and alternation procedures have failed to observe a disruption of spontaneous alternation by cortically ablated rats. It is possible that the testing of the animals under conditions of food deprivation was responsible for the deficit in this experiment. The penicillin-induced lesions failed to produce any noticeable change in the behavior of our subjects. Previous work had found behavioral changes in the acquisition of a two-way active avoidance task [16, 18], perhaps it is not surprising that the deficit demonstrated in these two studies was not found in the present study, as both the type of task
employed and the number of trials given each day were different. Penicillin-induced lesions of the hippocampus seem to have little effect on the learning of an appetitively motivated operant discrimination task [24] and this may mean there is little reason to assume that the acquisition of an appetitive task will be affected by brain damage in the same way as the acquisition of an aversive task. However, before we can conclude that the penicillininduced focus produces effects specific to avoidance tasks, two additional variables must be considered. First, the penicillin used in the present study (S-R Parke-Davis) had not been manufactured for several years. Hamilton [3] found that this penicillin was not generally effective in producing a behavioral deficit in the active avoidance task in an experiment conducted at about the same time as this study. Furthermore, the epileptogenic properties of a penicillininduced lesion may become less over time. In prolonged experiments like this and the operant discrimination task used by Woodruff and lsaacson [24] the postoperative experiences were continued over a prolonged period of time. However, as indicated in Fig. 3, abnormalities were found in the post-experiment EEGs of several animals with penicillininduced lesions. These results, as well as those of Schmaltz [18], Olton [16], and Hamilton [3], indicate that the need for further research into the physiological mechanisms underlying the behavioral effects of irritative lesions still is apparent.
REFERENCES 1. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-442, 1967. 2. Douglas, R. J. and R. L. lsaacson. Hippocampal lesions and activity. Psychonom. ScL 1: 187-188, 1964. 3. Hamilton, G. L. Effects of penicillin-induced epileptogenic foci in the hippocampus. Unpublished dissertation, University of Florida, 1970. 4. Hirsch, R. Lack of variability or perseveration: describing the effect of hippocampal ablation. Physiol. Behav. 5: 12491254, 1970. 5. lsaacson, R. L., R. J. Douglas and R. Y. Moore. The effect of radical hippocampal ablation on acquisition of an avoidance response. J. comp. physiol. Psychol. 54: 625-628, 1961. 6. Isaacson, R. L., H. Schwartz, N. Persoff and L. Pinson. The role of the corpus callosum in the establishment of areas of secondary epileptiform activity. Epilepsia 12: 133-146, 1971. 7. Kimble, D. P. and R. Kimble. Hippocampectomy and response perseveration in the rat. J. comp. physiol. Psychol. 60: 474-476, 1965. 8. Kimble, D. P. and R. Kimble. The effect of hippocampal lesions on extinction and hypothesis behavior in rats. Physiol. Behav. 5: 735-738, 1970. 9. Means, L. W. Cue utilization and perseveration in the hippocampectomized rat. Unpublished dissertation. Claremont Graduate School, 1961. 10. Means, L. W. and R. J. Douglas. Effects of hippocampal lesions on cue utilization in spatial discrimination in rats. J. comp. physiol. Psychol. 73: 254-260, 1970. 11. Means, L. W., J. D. Leander and R. L. Isaacson. The effects of hippocampectomy on alternation behavior and response to novelty. Physiol. Behav. 6: 17-22, 1971. 12. Means, L. W. and D. W. Walker. Unpublished observation.
13. Milner, B. Amnesia following operation of the temporal lobes. In: Amnesia, edited by C. W. M. Whitly and O. L. Zangwill. London: Butterworths, 1966. 14. Miiner, B. Disorders of memory after brain lesions in man. Neuropsychologia 6: 175-179, 1968. 15. Niki, H. Response perseveration following the hippocampal ablation in the rat. dap. psychol. Res. 8: I-9, 1966. 16. Olton, D. S. Specific deficits in active avoidance behavior following penicillin injection into hippocampus. Physiol. Behav. 5: 957-963, 1970. 17. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 18. Schmaltz, L. W. Deficit in active avoidance learning in rats following penicillin injection into hippocampus. Physiol. Behav. 6: 667-674, 1971. 19. Silviera, J. M. and D. P. Kimble. Brightness discrimination and reversal in hippocampally lesioned rats. Physiol. Behav. 3: 625-630, 1968. 20. Thomas, G. J. Maze retention by rats with hippocamapal lesions and with fornicotomies. J. comp. physiol. Psychol. 75: 41--49, 1971. 21. Walker, D. W., L. G. Messer and L. W. Means. Effects of ITI on go/no-go performance after hippocampal lesions in rats. Psychonom. Sci. 21: 285, 1970. 22. Walker, D. W., L. G. Messer, G. Freund and L. W. Means. The effect of ITI on single alternation performance in the hippocampectomized rat, in preparation. 23. Wirier, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962. 24. Woodruff, L. and R. L. Isaac.son. The effect of irritative and non-irritative hippocampal lesions on two behavioral tasks. 1971. Paper presented at the 79th annual convention of the American Psychological Association.
The Effect of a Twenty-four Hour Intertrial Interval on The Acquisition of Spatial Discrimination by Hippocampally Damaged Rats I L A R R Y W. M E A N S L M I C H A E L
L. W O O D R U F F s A N D R O B E R T L, I S A A C S O N
Department o f Psychology, University of Florida, Gainesville, Florida, 32601, U.S.A. (Received 18 A u g u s t 1971) MEANS, L. W., M. L. WOODRUFFAND R. L. ISAACSON.The effect of a twenty-four hour intertrial interval on the acquisition of a spatial discrimination by hippocampally damaged rats. PHYSIOL.BEHAV. g (3) 457--462, 1972.--Rats with either
aspiration- or penicillin-induced (irritative) lesions of the hippocampus or overlying neocortex and normal rats were trained on a spatial discrimination in a T-maze after being tested for spontaneous alternation. Animals were given only one trial per day (24 hr ITI). Only the animals with aspiration lesions of the hippocampus were impaired in acquiring the spatial discrimination. Animals with either hippocampal or neocortical lesions alternated at reduced rates. Hippocampal lesions Neocortical lesions Twenty-four hour intertrial interval
Penicillin-induced epileptogenic foci
THE PRESENT study was undertaken to determine the effects of aspirative and irritative lesions of the hippocampus on acquisition of an appetitive task. The experiments of Olton [16] and Schmaltz [18] have suggested that the discrepancies observed between some of the behavioral effects produced in certain epileptic patients by temporal lobe destruction [13, 14] and those produced in animals by experimental destruction of the hippocampus [1] might be due to the epileptic condition of the brains of the former. In their studies with animals with penicillin injected into hippocampus, Olton and Schmaltz found electrographic signs of epileptiform activity and also demonstrated impairments in the acquisition of a two-way active avoidance problem. Animals with radical bilateral hippocampal destruction, on the other hand, are known to acquire the two-way active avoidance task more rapidly than normal animals [5]. The results of these studies led us to believe that it was important to compare the effects of the two types of lesions in other types of tasks. The task selected for this experiment, a spatial discrimination, is one in which hippocampally ablated animals perform in the same manner as normal animals [7, 15], provided the intertrial interval (ITI) does not exceed 20--30 min [9, 10]. Since the intertrial interval (ITI) is influential in determining the behavior of animals with hippocampal destruction, we attempted to maximize this aspect of the training with a
Spatial discrimination
24 hr 1TI, i.e., giving one trial per day. In addition, since it has recently been shown that rats with hippocampal lesions use fewer strategies of hypotheses than normal rats in acquiring brightness discrimination [8, 19], it was decided to examine carefully the choice behavior of the animals during acquisition. Finally, we wanted to determine the effect of the penicillin-induced irritative lesions on spontaneous alternation. Accordingly, all animals were tested for spontaneous alternation prior to spatial discrimination learning. METHOD
Animals Fifty Long-Evans male rats, weighing between 304 and 413 g at the time of surgery, were used. The animals were housed individually under conditions of continuous light.
Surgery The animals were divided into 5 groups of 10 animals each. The hippocampal aspiration group (HA) received bilateral aspiration lesions of the hippocampus, as well as of the overlying neocortex, while the animals in the cortical aspiration group (CA) received ablations of only the neocortex overlying the hippocampus. The aspiration procedure employed has been described previously [5]. The hippocampal
1Supported in part by NIMH Grant 16384-03 to R. L. Isaacson and USPHS training grant MH-10320 to Center for Neurobiological Sciences, University of Florida Medical Center. The authors wish to thank Mrs. Pauletta Sanders for her excellent preparation of histological materials. SNow at Department of Psychology, East Carolina University, Greenville, North Carolina, 27834. 3Requests for reprints should be sent to Michael Woodruff, Department of Psychology, University of Florida, Gainesville, Florida, 32601. 457
458 penicillin group (HP) received bilateral implants of penicillin (Parke, Davis and Co.; Type S-R) in the hippocampus and the cortical penicillin group (CP) received similar bilateral penicillin implants in the overlying cortex. The penicillin implant procedure has been described by Olton [16]. The stereotaxic coordinates, used for both implant groups, were anterior 1.8 and lateral 5.1 [17]. The penicillin was placed 4.9 and 0.8 mm below the surface of the cortex in the HP and CP groups, respectively. All surgery was performed under asceptic conditions using sodium pentobarbital anesthesia (50 mg/kg). The control animals (group N) received no lesions.
Apparatus All behavioral testing was conducted in a wood T-maze painted a fiat gray. It consisted of a 8 × 4 in startbox, a 4 × 10 in runway, and two 4 × 18 in arms. All sections were 6 in deep and covered with k in mesh hardware cloth. Guillotine doors separated the startbox from the runway and the arms from the choicepoint. The tops of both choicearms of the maze were covered with cardboard.
MEANS, WOODRUFF AND ISAACSON seizure-like discharges which resemble those found after the application of penicillin to neural tissue [6]. The recording sessions began 20 min following the lowering of the electrodes. Olton [16] and Schmaltz [18] have observed injury spikes as a result of placing the electrodes in the hippocampus, but these spikes continue only a few minutes and are easily differentiated from the spike activity produced by penicillin.
Histology After completion of behavioral and electrophysiological testing, all operated animals were sacrificed and intracardially perfused with 0.9 per cent saline and I0 per cent formalin. The brains were removed, embedded in celloidin, and sectioned at 30 ~t. Every tenth section of the aspiration animals' brains and every second section of the brains of the penicillinimplanted animals was stained with thionin and examined for the locus of the drug implant or the extent of the lesion.
RESULTS
Histology Procedure Preliminary handling and training was begun 11 days preoperatively. On the first 3 days of this period all animals were handled in groups of five to seven on a table for a period of 30 min. The deprivation schedule was initiated on Day 3. Except for the day prior to surgery and for 14 days postoperatively, all animals were maintained at 8 5 ~ of their ad lib body weight throughout the experiment. They were weighed and fed immediately following their daily handling or test session. On Days 5-7 of the preoperative period each rat was handled individually and exposed to Noyes 0.045 g food pellets placed in a small dish on top of the handling table. On days eight and nine preoperatively, all rats were allowed to individually explore the T-maze for 5 rain, or until the 4 Noyes pellets placed in the food cup of each arm were consumed, whichever occurred first. Spontaneous alternation testing was conducted on the 14th to 16th postoperative days. All rats were run each day until they completed six trials or until they failed to make a choice within 5 min of being placed in the maze. Upon making a choice, the animals was retained in the goal box for 20 sec and then given another trial. Food pellets were not present in the maze during spontaneous alternation training. On postoperative Days 17 and 18 four 0.045 g Noyes food pellets were placed in both choice-arms, and the rats were allowed to individually explore the maze for 5 rain, or until they consumed all the pellets, whichever came first. Spatial discrimination training was initiated on postoperative Day 19. Each rat was given one non-correction trial each day. All rats were trained to their nonpreferred side, namely, that side which was chosen least frequently on the first trial of each of the three alternation days. Four 0.045 g Noyes pellets constituted the reinforcement for a correct choice. All rats were run until they made a correct response on 10 consecutive trials. Upon completion of behavioral testing bipolar electroencephalographic recordings were made from the sites at which penicillin had been placed in seven of the HP and five of the CP animals. All recordings were done with the animals placed in a stereotaxic instrument while anesthetized with a 1 5 ~ urethane solution (1.75 g/kg, IP). Urethane, in the amounts used in this experiment, does not in itself produce
Examination of the brain sections revealed that animals in the hippocampal lesion group had undergone almost total removal of the dorsal and lateral hippocampus; various amounts of the ventral hippocampus were spared in different animals. The neocortex and capsule fibers overlying the hippocampus were destroyed bilaterally. The posterior edge of the lesion typically extended into the entorhinal cortex. Slight unilateral thalamic damage was found in three of the ten subjects. In five animals of this group bilateral degeneration of portions of the dorsal lateral geniculate nucleus of thalamus, as well as the lateral and posterior thalamic nuclei was observed. No behavioral effects could be correlated with the position, or presence, of thalamic degeneration. No thalamic degeneration was observed in the cortically ablated animals. The degeneration could possibly be due to involvement of the optic radiations in the course of removing the hippocampus. Examination of the lesions produced in the cortical group revealed that an amount of neocortex comparable to that removed in the hippocampal ablations had been removed. One animal in this group was an exception in that unilateral hippocampal destruction was found. This animal and one other animal from this group that died during the course of the experiment were eliminated from the data analysis. In the penicillin-implanted animals microscopic examination revealed that the penicillin produced small lesions of about 1 mm in dia. Reconstructions of the maximum and minimum extent of the aspirative lesions and the placements of the penicillin are presented in Fig. 1. Figure 2 shows photomicrographs of representative neocortical and hippocampal penicillin implants.
Eleetroencephalographic Results Abnormal electrical activity was recorded from the site of drug application in five of the seven animals from which recordings were made with hippocampal placements and from three of the five animals with neocortical placements from which recordings were made. The abnormal activity, recorded at the end of training, differed from that observed immediately following penicillin implant, when sharp spikelike activity appeared against a depressed background. Figure 3 shows the representative E E G from a pilot animal
FIG. 2. Photomicrographs of representative cortical (upper) and hippocampal (lower) penicillin lesions.
(facing page 458)
HIPPOCAMPAL LESIONS AND INTERTRIAL INTERVAL
459
FIG. 1. Minimum (darkened area) and maximum (cross-hatched) lesions extents for group CA (upper left two rows) and group HA (lower left two rows). Penicillin placements for group CP (upper right two rows) and group HP (lower right two rows).
immediately following placement of penicillin in the hippocampus (bottom 2 traces). The records from one hippocampally-implanted animal at the end of training showed no sharp waves in the EEG. Two of the hippocampallyimplanted animals and one cortically-implanted animal that did show spike-activity are also included in this figure. There was, however, no correlation between the presence or absence of epileptiform activity and the performance of an animal on the behavioral tests utilized in this study.
Behavioral Results The mean alternation rate of five groups is shown in Table 1. In agreement with the findings of Douglas and Isaacson [2] and Means, Leander, and Isaacson [11], the H A group made significantly fewer spontaneous alternations than did the N, CP, or HP groups (one-way analysis of variance F - - 7.68, dr= 4,42, p < 0.01). However, an a posteriori Newman-Keuls analysis [23] indicated that both the CA and the H A group alternated at a significantly lower rate than the other three groups (p < 0.01 in all cases). F o r the analysis of the spatial discrimination task, the number of trials for each rat to reach successive criteria of eight correct responses in 10 trials, nine correct responses in 10 trials, and 10 correct responses in 10 trials were examined (Table 1). A one-way analysis o f variance indicated a significant difference on both the 8/10 and 9/10 criterion measures (F ~ 6.76, d f = 4,43, p < 0.01 ; F ---- 3.49, d f ~ 4,43, p < 0.05), but no differences were found using the 10/10 criterion. A Newman-Keuls analysis was performed on the data of the two significant criteria and the H A group was found to be significantly different (p < 0.01) from the other four groups, which did not differ from each other. Further analysis of the data indicated that the H A subjects made significantly more errors before reaching the terminal criterion of 10 correct responses out of 10 trials than did the CA, N, HP, or CP groups (F ~- 2.96, d f = 4,43, p < 0.05). Figure 4 presents an analysis of the frequency with which sequences of consecutive responses were made to the rewarded (defined as originally non-preferred) and non-rewarded (defined as originally preferred) sides of the T-maze. The ten consecutive rewarded responses which comprise the
terminal criterion are omitted from this analysis. Except the group suffering hippocampal lesions, all groups exhibited very similar patterns of response sequences. The number of sequences of one response to either the rewarded or nonrewarded side of the maze were essentially equal for the hippocampally lesioned animals, while the other groups made significantly more single responses to the non-rewarded (preferred) side. Chains of responses of more than two in a row were made most frequently to the rewarded arm of the maze by all groups except the hippocampally ablated group, which exhibited more sequences of responses to the rewarded non-preferred arm only for sequences of three or more consecutive responses. It should also be noted that the hippocampally lesioned animals differed from the other groups in that seven of the ten animals in this group exhibited long chains of responses to the preferred arm of the maze. However, the animals with hippocampal destruction which did not exhibit long strings of incorrect responses learned the same problem as quickly and exhibited the same response patterns as did animals in the other groups of subjects. Examinations of the behavioral results (Table 1) indicates that neither the HP or CP group differed from group N, the normal control group, on either spontaneous alternation or spatial discrimination. Analysis of variance and subsequent Newman-Keuls confirm these observations. Groups HP, CP, and N were not found to differ on any of the behavioral tests. DISCUSSION
Animals in group H A took significantly more trials to attain the 8/10 and 9/10 criteria and made significantly more errors before reaching the terminal criterion of 10/I0 than all other groups tested, clearly indicating that hippocampal ablation produced a deficit in the acquisition of this task. Other studies examining the effects of hippocampal lesions on spatial discrimination have failed to observe a deficit with short intertrial intervals [7, 9, 15]. However, Means and Douglas [10] observed a deficit in hippocampally ablated rats in a situation where most of the animals were trained to their non-preferred side. Kimble and Kimble [7] also trained their animals to the non-preferred side and failed to
460
MEANS, WOODRUFF AND ISAACSON TABLE 1 MEAN PER CENT SPONTANEOUS ALTERNATION AND MEAN NUMBER OF TRIALS TO SUCCESSIVE CRITERIA ON SPATIAL DISCRIMINATION
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observe a deficit on the original acquisition of a spatial discrimination in a Y-maze. These latter authors determined the animals' non-preferred arm of the maze in quite a different manner from that used in the present study. They determined the non-preferred arm on the basis of the responses made on the first five trials given on the first training day. Rewards were present in both arms of the maze on these trials. Formal training was begun on trial six. Therefore, we cannot exclude the possibility that the training to the non-preferred side, measured by the first response made each of three days during the spontaneous alternation tests, was a factor in the present experiment. The situation is further confounded by the fact that Means and Douglas [10] gave their four preacquisition test trials during which the first response made by the animal
Spontaneous Alternation Spatial Discrimination
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48 15.6 17.3 26.4
81 15.5 21.I 40.5
78 17.5 28.2 39.6
84 12.6 21.0 36.5
was reinforced. Kimble and Kimble [7] reinforced responses to both arms of the maze when determining the animals' preferences. The present study did not differentially reinforce the animals prior to acquisition training. The present study differs from previous studies in that it employed a much longer ITI, 24 hr. The longest ITI of which we are aware that has been employed with hippocampally ablated rats is 30 min [10]. The second possible explanation of the present results is that the deficit stems from the long intertrial interval used. Such an explanation would be consonant with the results reported from the study of an epileptic patient who has had the mesial portions of the temporal lobes resected [13]. This patient can remember a series of digits for several minutes if not disrupted. If a disrupting stimulus, such as a comment by the experimenter occurs, causing a shift in attention, the patient forgets the digits as well as the retention task itself. Possibly the longer ITI allows more opportunity for disrupting stimuli to produce attention shifts and subsequent memory deficits. Consistent with this hypothesis, hippocampally ablated rats have been shown to be deficient on a single alternation task when ITIs longer than 20 sec are employed [21, 22] or when another problem is introduced during the ITI [12]. Also, Hirsch [4] has recently suggested employing a memory hypothesis to explain reduced variability by hippocampectomized rats in a four choice situation. One difficulty with a simple hypothesis based upon an enhanced rate of memory loss over time is the fact that the animals with aspirative lesions of the hippocampus do not choose randomly between the arms of the maze. If each response was made without regard to the previous response(s), i.e., with no memory of the past at all, the long sequences of ten and more responses, which mark the performances of most animals with hippocampal damage, made to the preferred side of the maze would be difficult to explain (see Fig. 4). Moreover, it is obvious that animals with hippocampal lesions do retain information from one day to the next. Animals trained in a spatial discrimination task with shorter ITIs which acquire the problem carry over the information from day to day. Therefore, if the effect of the hippocampal lesion is to disrupt retention over extended time-periods then this deficit is specific to a certain kind of information related to the reward or non-reward of a response and not about the solution to the problem. The analysis of the frequency of correct and incorrect response sequences was undertaken to reveal any differences between the impaired performance of subjects with hippocampal lesions and the performance of animals in other
HIPPOCAMPAL LESIONS AND INTERTRIAL INTERVAL
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groups. We believed that comparing sequences to the preferred and non-preferred sides would enable us to evaluate the possibility that animals with hippocampal lesions had a side, position, or response preference. All of these were confounded in the present experiment and such preferences might have been more difficult to overcome for hippocampally ablated animals than for other animals. We reasoned that this preference could be expressed as an altered probability of response to the two arms of the maze in the presolution period. Widely discrepant response probabilities should be revealed in widely different frequencies of occurrence of strings of consecutive responses of different length, assuming independence of responses. For example, if the probability response to the right arm was 0.8 then the probability of making two consecutive responses to the right arm would be 0.64, while the probability of two consecutive responses to the left arm would be only 0.04. If the probability of response to the two arms was equal, the probability of two consecutive responses to either arm would be 0.25. There was some reason to believe that animals with hippocampal lesions might have a different or
stronger side preference since all but one animal in this group had a preference for the right arm of the maze while animals in other groups distributed their preferences more evenly between the two arms. However, no evidence for a differential tendency to respond to the preferred arm was found for these animals, and in fact their single responses were more equally divided among the preferred and nonpreferred arms of the maze than by animals in other groups. The fact that all animals made more consecutive responses to the non-preferred (rewarded) arm than the preferred (non-reward) arm in the pre-solution period suggests that reward influences the animals' behavior before the beginning of the criterion block of trials and that this compensates for any preferential tendency to respond to the preferred side. The group of animals with hippocampal lesions differed from the other groups in that animals in this group were more 5kely to make long series of incorrect responses, always to the preferred side. There were animals in this group, however, that were unimpaired in acquiring the problem and produced behavior similar to that of the other groups. It becomes apparent, then, that the behavioral impairment
462
MEANS, WOODRUFF AND 1SAACSON
produced by the lesion was made manifest by a greater likelihood that individual animals would make one or more long sequences of incorrect responses. The effect was all-ornone. Either the animal made a long sequence of errors (10 or more) or it did not. The frequency of response sequences of intermittent length is no different than that found in other groups of animals. This observation of all-or-none behavioral deficits among a group of animals suffering from hippocampal lesions has recently been emphasized by Thomas [20]. The finding that hippocampally ablated rats lack the normal tendency of a rat to spontaneously alternate has often been reported in the literature, but the results of the present study indicate that cortically lesioned animals may also alternate significantly less than normals. It should be noted, however, that other studies employing similar cortical lesions and alternation procedures have failed to observe a disruption of spontaneous alternation by cortically ablated rats. It is possible that the testing of the animals under conditions of food deprivation was responsible for the deficit in this experiment. The penicillin-induced lesions failed to produce any noticeable change in the behavior of our subjects. Previous work had found behavioral changes in the acquisition of a two-way active avoidance task [16, 18], perhaps it is not surprising that the deficit demonstrated in these two studies was not found in the present study, as both the type of task
employed and the number of trials given each day were different. Penicillin-induced lesions of the hippocampus seem to have little effect on the learning of an appetitively motivated operant discrimination task [24] and this may mean there is little reason to assume that the acquisition of an appetitive task will be affected by brain damage in the same way as the acquisition of an aversive task. However, before we can conclude that the penicillininduced focus produces effects specific to avoidance tasks, two additional variables must be considered. First, the penicillin used in the present study (S-R Parke-Davis) had not been manufactured for several years. Hamilton [3] found that this penicillin was not generally effective in producing a behavioral deficit in the active avoidance task in an experiment conducted at about the same time as this study. Furthermore, the epileptogenic properties of a penicillininduced lesion may become less over time. In prolonged experiments like this and the operant discrimination task used by Woodruff and lsaacson [24] the postoperative experiences were continued over a prolonged period of time. However, as indicated in Fig. 3, abnormalities were found in the post-experiment EEGs of several animals with penicillininduced lesions. These results, as well as those of Schmaltz [18], Olton [16], and Hamilton [3], indicate that the need for further research into the physiological mechanisms underlying the behavioral effects of irritative lesions still is apparent.
REFERENCES 1. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-442, 1967. 2. Douglas, R. J. and R. L. lsaacson. Hippocampal lesions and activity. Psychonom. ScL 1: 187-188, 1964. 3. Hamilton, G. L. Effects of penicillin-induced epileptogenic foci in the hippocampus. Unpublished dissertation, University of Florida, 1970. 4. Hirsch, R. Lack of variability or perseveration: describing the effect of hippocampal ablation. Physiol. Behav. 5: 12491254, 1970. 5. lsaacson, R. L., R. J. Douglas and R. Y. Moore. The effect of radical hippocampal ablation on acquisition of an avoidance response. J. comp. physiol. Psychol. 54: 625-628, 1961. 6. Isaacson, R. L., H. Schwartz, N. Persoff and L. Pinson. The role of the corpus callosum in the establishment of areas of secondary epileptiform activity. Epilepsia 12: 133-146, 1971. 7. Kimble, D. P. and R. Kimble. Hippocampectomy and response perseveration in the rat. J. comp. physiol. Psychol. 60: 474-476, 1965. 8. Kimble, D. P. and R. Kimble. The effect of hippocampal lesions on extinction and hypothesis behavior in rats. Physiol. Behav. 5: 735-738, 1970. 9. Means, L. W. Cue utilization and perseveration in the hippocampectomized rat. Unpublished dissertation. Claremont Graduate School, 1961. 10. Means, L. W. and R. J. Douglas. Effects of hippocampal lesions on cue utilization in spatial discrimination in rats. J. comp. physiol. Psychol. 73: 254-260, 1970. 11. Means, L. W., J. D. Leander and R. L. Isaacson. The effects of hippocampectomy on alternation behavior and response to novelty. Physiol. Behav. 6: 17-22, 1971. 12. Means, L. W. and D. W. Walker. Unpublished observation.
13. Milner, B. Amnesia following operation of the temporal lobes. In: Amnesia, edited by C. W. M. Whitly and O. L. Zangwill. London: Butterworths, 1966. 14. Miiner, B. Disorders of memory after brain lesions in man. Neuropsychologia 6: 175-179, 1968. 15. Niki, H. Response perseveration following the hippocampal ablation in the rat. dap. psychol. Res. 8: I-9, 1966. 16. Olton, D. S. Specific deficits in active avoidance behavior following penicillin injection into hippocampus. Physiol. Behav. 5: 957-963, 1970. 17. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 18. Schmaltz, L. W. Deficit in active avoidance learning in rats following penicillin injection into hippocampus. Physiol. Behav. 6: 667-674, 1971. 19. Silviera, J. M. and D. P. Kimble. Brightness discrimination and reversal in hippocampally lesioned rats. Physiol. Behav. 3: 625-630, 1968. 20. Thomas, G. J. Maze retention by rats with hippocamapal lesions and with fornicotomies. J. comp. physiol. Psychol. 75: 41--49, 1971. 21. Walker, D. W., L. G. Messer and L. W. Means. Effects of ITI on go/no-go performance after hippocampal lesions in rats. Psychonom. Sci. 21: 285, 1970. 22. Walker, D. W., L. G. Messer, G. Freund and L. W. Means. The effect of ITI on single alternation performance in the hippocampectomized rat, in preparation. 23. Wirier, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962. 24. Woodruff, L. and R. L. Isaac.son. The effect of irritative and non-irritative hippocampal lesions on two behavioral tasks. 1971. Paper presented at the 79th annual convention of the American Psychological Association.