Hippocampal lesions alter conditioning to conditional and contextual stimuli

Behavioural Brain Research 88 (1997) 219 – 229

Research report

Hippocampal lesions alter conditioning to conditional and contextual stimuli Gordon Winocur * Rotman Research Institute, Baycrest Center for Geriatric Care, 3560 Bathurst Street, Toronto, Ontario M6A 2E1, Canada Department of Psychology, Trent Uni6ersity, Toronto, Ontario M61 2E1, Canada Departments of Psychology and Psychiatry, Uni6ersity of Toronto, Toronto, Ontario M61 2E1, Canada Received 17 January 1997; received in revised form 25 February 1997; accepted 25 February 1997

Abstract The control of conditioned fear behaviour by a conditional stimulus (CS) and contextual stimuli (CXT) was compared in rats with lesions to the hippocampus (HPC) or neocortex (CO), and operated controls (OC). After classical fear conditioning in a distinctive context, rats were subsequently tested in the presence of the CS and CXT (CS + CXT), the CS alone (CS-only), or context alone (CXT-only). Two experiments were conducted in which conditioned fear was measured by an active avoidance response (experiment 1) or by response suppression (experiment 2). Groups did not differ in acquiring the conditioned fear response, as measured in the CS +CON test but, in both experiments, hippocampal (HPC) groups exhibited more conditioned fear behaviour than controls in the CXT-Only and CS-Only conditions. It was suggested that control rats conditioned the fear response to a stimulus complex that incorporated the CS and CTX. Rats with HPC lesions did not form this association between the stimulus elements; instead they segregated the CS and CXT and formed independent associations between the conditioned response (CR) and each component. In showing that HPC damage disrupts the process of forming associations between environmental stimuli and that the effect is not restricted to contextual cues, the results help to resolve apparently contradictory findings regarding the role of HPC in contextual information processing. © 1997 Elsevier Science B.V. Keywords: Hippocampus; Contextual cues; Fear conditioning

1. Introduction In a previous study, Winocur et al. [38] assessed the effects of hippocampal (HPC) lesions in rats on classical fear conditoning to background or contextual stimuli. In control groups, conditioning to context was inversely related to the probability that the conditional stimulus (CS)(tone or light) predicted the US (foot shock). In contrast to the graded contextual conditioning displayed by the controls, HPC groups showed strong conditioning to context regardless of the * Tel.: +1 416 7852500, ext. 3592; fax: + 1 416 7852862; e-mail: [email protected] 0166-4328/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 4 3 2 8 ( 9 7 ) 0 0 0 4 5 - 4

relationship between CS and US. These results are consistent with other evidence that animals with HPC damage form unusually strong associations between learned responses and contextual cues, and that these cues exert abnormal control over the animals’ behaviour [14,25,29,35,37]. This effect of HPC lesions can be attributed to a processing deficit that prevented the formation of relationships between stimuli that are associated with a reinforcing event. Failure of this associative mechanism results in inefficient learning strategies that can lead to separate conditioning to discrete CSs and contextual stimuli (CXT) that normally would be integrated as part of a stimulus complex. This view is in accord with

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theories of HPC function that emphasize the structure’s role in forming relationships between stimuli [6,28] and, further, predicts that HPC dysfunction leads to an abnormal distribution of associative strength among cues present during conditioning. In another study, Penick and Solomon [23] studied the relationship between hippocampus, context, and conditioning of the nictitating membrane response (NMR) in rabbits to a tone CS. HPC-lesioned rabbits normally acquired the conditoned NMR and were subsequently tested in either the same or a distinctly different context. A sharp decrease in the number of conditioned responses (CRs) to the CS was observed in control groups that were switched to the new context. In contrast, HPC groups continued to respond to the CS at a high level in both contexts. These results showed that rabbits with HPC lesions respond differently to context during classical conditioning. Similar results were obtained by Honey and Good ([11]; experiment 1) when rats with HPC lesions acquired an appetitive CR in one context and were then presented with the same CS in a different context. These data suggest that, in operant conditioning situations, HPC animals tend to segregate environmental stimuli and form strong associations with the CS that are independent of any conditoning to contextual cues. In a sense, Penick and Solomon [22] and Honey and Good [11] demonstrated the inverse effect of the Winocur et al. [38] study. In both sets of experiments, classical conditioning took place in a distinctive context. At test, Winocur et al. [38] presented the CXT in the absence of the CS, whereas Penick and Solomon [23] and Honey and Good [11] presented the CS in a totally different context. In both cases, HPC groups, but not controls, continued to perform the CR. None of the studies assessed conditioning strength in both test conditions, that is, with only CS or only CXT present. Although animals with HPC lesions form associations between CXT and CRs, they are impaired in tests where contextual cues are used to discriminate between conflicting responses that are paired with different stimuli [7,9,27]. The latter results have been attributed to a failure to use contextual information, although other evidence clearly indicates that contextual cues can be used as discriminative stimuli in conditional learning tasks [7,13,27] Another interpretation of the observed deficits is that HPC and control rats adopted different strategies in solving the multiple-cue conditional learning tasks. According to this view, controls combined the stimulus components into a unified stimulus that permitted efficient discrimination learning. By comparison, animals with HPC damage, unable to form the appropriate associations, segregated the various stimuli and proceeded to form direct relationships between each stimulus component and the corresponding response.

The purpose of the present study was to clarify the response of rats with hippocampal lesions to task-related stimuli in a test of classical fear conditioning. Following training, in which the CS (tone) and contextual stimuli (black compartment) are both reliably associated with the US (foot shock), fear behaviour was tested in the same context but without the CS present, or in response to the CS presented in a different context. It was predicted that, during acquisition, normal rats will combine the relevant stimuli into a single entity and condition the response to the stimulus complex as a whole. Removal of a significant component, either the CS or the CXT, should lead to reduced responding in these rats. On the other hand, because of their inability to form a conditional relationship involving a CS and CXT, it was predicted that HPC rats would form separate associations between the CR and the CS, and between the CR and CXT. Removing either component from the stimulus complex should produce a smaller response decrement in these animals.

2. Experiment 1

2.1. Method 2.1.1. Subjects The subjects for this experiment were 68 naive, male, Long-Evans rats, approximately 6 months old. The rats were obtained from the Trent University Breeding Centre and housed individually in wire cages, with food and water available at all times. 2.1.2. Surgery and histology The rats were anesthetized with sodium pentobarbital and positioned in a Johnson-Krieg stereotaxic instrument with the tooth bar raised to a height of 5 mm above the interaural plane. Lesions were produced electrolytically by passing a 2 mA direct current for 15 s through a stainless steel electrode that was insulated except for 0.5 mm at the tip. HPC lesions were produced bilaterally at 2.2 mm posterior to Bregma, at a depth of 3.0 mm below the dural membrane, and at 1.5 and 2.5 mm lateral to the midline. The same A-P and lateral coordinates were used to make cortical (CO) lesions but the electrode was lowered only 1.5 mm below dura. The rats that served as operated controls (OC) were anesthetized, holes were drilled in their skulls, but no electrode was lowered. After the experiment, rats with HPC or CO lesions were deeply anesthetized and perfused through the heart with physiological saline followed by 10% formol saline. Frozen sections of the damaged areas were made in the tranverse plane and every fifth section was stained with thionin. Locus and extent of brain damage

G. Winocur / Beha6ioural Brain Research 88 (1997) 219–229

were assessed by comparison with the standard atlas of Ko¨nig and Klippel [16]

2.1.3. Apparatus All rats were trained initially in a wood box that was divided into a black (20×16 × 18-cm) compartment and a white (44×25 ×18-cm) compartment (box 1). An opaque Plexiglas sliding door separated the two compartments. The box was covered by a hinged roof of clear Plexiglas with numerous holes to allow ventilation. The floor consisted of metal rods, spaced 1.3 cm apart. A second wood box (50×40 × 18-cm)(box 2), made entirely of wood, was divided into equal sized left and right compartments (box 2). The inside walls and floor of box 2 were stained with a clear finish. A third box (30 × 30 ×20-cm), constructed of clear Plexiglas with a floor made of metal rods (box 3), was located in a different room. The boxes were placed on a table, 1.3 m above the floor. Training and testing were conducted in a quiet room in dim illumination that was controlled by a rheostat. The CS was an 80 db tone presented through a speaker that was centrally mounted about 10 cm above either box. The US was a 1 s, 1.2 mA foot-shock delivered by a BRS/LVE shock generator (c SSG-003). 2.1.4. Procedure About 1 month after surgery, rats were handled daily in pairs for about 20 min on 5 consecutive days. On the following day, they were assigned to the CS +CXT (paired), CS+CXT (unpaired), CXT-Only, or the CSOnly condition. The number of rats assigned to the various surgical groups and experimental conditions are shown in Table 1. The experiment was conducted in three stages, with stage 1 identical for all groups: 2.1.4.1. Stage 1. The purpose of stage 1 was to familiarize the rats with both boxes and to establish their natural preferences for the compartments in each box. On day 1, each rat was placed, according to a random Table 1 Group numbers for all conditions in experiments 1 and 2 CS+CXT (P)

CS+CXT (U)

CXT-Only

CS-Only

8 8 8

8 7 8

9 8 8

10 8 8

Experiment 2 HPC 7 Control 8

8 8

8 8

7 7

Experiment HPC CO OC

1

P, Paired; U, Unpaired

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order, in the left or right compartment of box 2, facing away from the centre. The sliding door was removed and the rat was free to wander throughout the box. On day 2, the procedure was repeated except that each rat was placed in the other compartment of box 2. On day 3, each rat was placed in the white or black compartment of box 1, facing away from the centre. The sliding door was removed and the rat was free to explore both compartments. On day 4, the procedure was repeated except that each rat was placed in the other compartment. Once again, the order of placement in the compartments was randomly determined. Each session lasted 300 s and the amount of time spent in both compartments was recorded.

2.1.4.2. Stage 2. This stage was initiated on day 5 and was identical for all groups except the CS+ CXT (unpaired) group. For the three groups that received CSUS pairings, each rat was placed individually in the black compartment of box 1 with the sliding door closed to prevent access to the white compartment. After about 5 min, a series of CS (tone) and US (foot shock) paired trials was initiated. For each pairing, the tone was presented for 10 s, along with a 1.2 mA foot shock which was delivered just before the end of the CS period. Termination of the shock coincided with the end of the tone. Ten such pairings were administered, separated by a variable interval that ranged between 18 and 178 s, and averaged 98 s. After the last CS-US pairing, the rat remained in the black compartment for an additional 5 min during which neither the CS nor the US was administered. Rats in the CS+ CXT (unpaired) group were placed in the black compartment and received ten presentations of the CS alone according to the same schedule as the other groups. Several hours later, they were placed in the Plexiglas box (box 3) and were administered ten, 1.2 mA foot shocks, following the same schedule. The purpose of this condition was to assess whether simply experiencing the CS and the US, in an unrelated way, affected behaviour during subsequent testing. 2.1.4.3. Stage 3. On day 8, the groups were tested as follows: 2.1.4.3.1. CS + CXT (paired and unpaired). Rats in these conditions were placed in the black compartment of box 1, facing away from the centre of the apparatus. The sliding door was in place to block immediate entry to the white compartment. After a few seconds, with the rat facing the centre, the CS was presented. Immediately after the 10 s CS, the sliding door was opened, and the rat allowed to enter the white compartment. In this, and the other testing conditions, the US was not administered. In all conditions, the rat was removed after 300 s had elapsed. Records were kept of each rat’s response latency and the amount of time spent in each compartment

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2.1.4.3.2. CXT-Only. Testing procedure was identical to that of the CS+ CXT conditions except that the CS was not administered. The sliding door was opened approximately 10 s after the rat had turned to face the centre of the apparatus. 2.1.4.3.3. CS-Only. Rats in this condition were tested in box 2. Each rat was placed in the right compartment, facing away from the centre. When the rat had turned towards the centre, the CS was presented for 10 s. The offset of the CS coincided with the opening of the door, allowing the rat to enter the other compartment.

3. Results Pre-training preferences for the compartments of both boxes were established by comparing the cumulative time spent in each compartment. There was no effect in either box related to the compartment in which rats were placed first. In box 1, the expected preference for the black compartment was observed as all groups spent 2 to 3 times longer in that compartment than in the white compartment. By comparison, rats divided their time equally between the left and right compartments of box 2. Analysis of variance (ANOVA) applied to the percentage time spent in the black compartment of box 1 and the left compartment of box 2 revealed no significant differences between lesion groups or between rats assigned to the various training/test conditions. HPC lesions did not affect acquisition of the CR. Table 1 shows that all groups displayed the same level of performance when tested in the CS+CXT (paired) condition. In that test, all groups exhibited short latencies to leave the black compartment and spent much less time in that compartment throughout the test session. ANOVA revealed no differences between groups in terms of latency, or time spent in the black compartment (both F’s B1). Table 1 also shows that none of the groups exhibited fear behaviour when tested in the CS +CXT (unpaired) condition. ANOVA confirmed non-significant group differences on the latency and cumulative time measures (F’sB 1). By comparison, the HPC groups exhibited more fear behaviour than the control groups in the other test conditions (see Table 2). In the CXT-Only test, where rats’ responsiveness to contextual cues alone was tested, the HPC group had shorter latencies and spent less time in the black compartment than the CO and OC groups. ANOVA revealed a significant group effect on the latency (F(2,22)=6.02, P B 0.01) and cumulative time, (F(2,22)= 8.03, P B 0.01), measures. The same pattern of results was obtained in the CS-Only test, conducted in box 2, where the HPC Group again had shorter response latencies (F(2,22) = 4.52, P B 0.025) and spent more time in the ‘safe’ compartment (F(2,22)= 4.01, PB 0.05) than the other groups. Post-

Table 2 Mean response latencies (RL) and cumulative time (CT) spent in the shock-associated compartment of experiment 1 (a)

CS+CXT Paired

Unpaired

HPC

CO

OC

HPC

CO

OC

RL (s) S.D. CT (% of total) S.D .

18.3 6.2 16.3 7.8

16.0 4.7 19.5 6.4

17.5 5.5 18.8 8.9

231.0 51.7 85.4 12.5

228.1 46.7 83.4 8.6

237.9 29.1 97.6 8.5

(b)

CS-Only

RL (s) S.D. CT (% of total) S.D.

30.7 14.0 17.8 7.4

73.9 26.8 40.0 5.7

CXT-Only 92.0 21.9 49.1 10.8

46.7 22.8 22.7 6.0

186.5 50.8 71.8 12.3

227.1 45.8 84.6 8.8

(a) Mean RLs and CTs spent in shock-associated black compartment during testing in CS+CXT (paired) and (unpaired) conditions of experiment 1. (b) Mean RLs and CTs spent in shock-associated compartment in CS-Only and CXT-Only conditions.

hoc comparisons with the Tukey test indicated that, in each test condition, the HPC group consistently differed from CO and OC groups on both measures (all P’sB 0.05), whereas the control groups did not differ significantly from each other (P’s\ 0.05). An overall ANOVA was conducted on the response latency data of the CS-Only and CXT-Only Groups to compare responses to the different cues. In addition to confirming the significant lesion effect (F(2,45)=5.52, PB 0.01), this analysis revealed a statistically significant cuing effect (F(1,45)=7.35, PB 0.01) and a significant group× cue interaction (F(2,45)=4.86, PB 0.05). Post-hoc analyses with the Tukey Test showed that all groups responded faster in the CS-Only Condition (P’sB 0.01). However, as can be seen in Table 2, the differences between the HPC Groups and the control groups were greater in the CXT-Only Condition. The same analysis performed on the cumulative time data revealed an identical pattern.

4. Discussion The results of experiment 1 show that, following training in which foot-shock is paired with a discrete CS and distinctive contextual cues, HPC and control groups displayed similar conditioned fear when tested in the presence of both stimuli. However, when only the CS or the context was present at test, the HPC group exhibited considerably more fear behaviour. This outcome points to important differences in the way that HPC and normal rats form relationships between environmental stimuli as part of the conditioning process.

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The finding that HPC lesions resulted in strong fear conditioning to context appears to be at variance with reports that rats with HPC lesions displayed abnormally weak contextual conditioning in similar paradigms [15,24,29]. This issue will be addressed in detail in Section 9, but there is one important procedural difference that could account for the discrepant results. In experiment 1, conditioned fear was measured by the rat’s active avoidance of the compartment associated with shock. During stage 1 training, rats learned that they could move freely between the compartments [21,38]. Accordingly, when the sliding door was removed at test, normal rats avoided a shock-associated area by quickly moving to the other compartment. In studies that yielded impaired fear conditioning to context following HPC lesions, fear was measured typically by response suppression. In those studies, a confinement procedure was used in which rats had no opportunity to escape during training or testing and, under such conditions, the dominant fear response to anticipated shock is freezing. The question arises as to whether the role of the HPC in conditioning to context is tied directly to the nature of the CR. Several authors have argued that HPC lesions exert a disinhibitory effect that results in reduced behavioural control in various experimental situations [4,8]. Following this view, rats with HPC lesions would have a lower threshold for activity than normal rats in the presence of shock-related cues. In the test conditions of experiment 1, lesion-induced increased activity would result in more time spent in the opposite compartment and give the impression that strong conditioning to context had occurred. In contrast, in the confinement procedure, where freezing is a more probable expression of conditioned fear, if HPC rats were more active in the presence of contextual cues that would suggest weaker conditioning. The second experiment addressed this issue by altering the procedures of experiment 1 and turning the test into a version of inhibitory avoidance. In inhibitory avoidance conditioning, suppression of a preferred response is the measure of conditioned fear. As in experiment 1, cue-shock pairing during fear conditioning took place in the small black compartment. However, to test for conditioning to context, rats were placed in the white compartment and fear was measured by latencies to move to the black compartment and the total time spent in that compartment. If HPC lesions selectively interfered with conditioning an inhibitory response to context, the HPC group should move readily to the black compartment and spend more time there than controls. On the other hand, if response disinhibition is not a factor and if HPC rats form separate associations between fear responses and salient environmental cues, as suggested by the results of ex-

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periment 1, HPC rats should show greater avoidance of contextual or conditional stimuli, whether the stimui are presented together or alone.

5. Experiment 2

5.1. Method Sixty-one naive, male, Long-Evans rats, approximately 6 months old, were subjects in this experiment. Of these, 30 received bilateral HPC lesions, 15 received CO lesions, and 16 comprised an OC group. Surgical and histological procedures were identical to those followed in experiment 1. A total of 3–4 weeks after surgery, each rat was assigned to one of four conditions–S + CXT (paired), CS+CXT (unpaired), CXT-Only, or CS-Only. Shaping and training were conducted in the same way as in experiment 1. The only departure from experiment 1 occured in the testing phase. In the test trials of the CS+CXT (paired and unpaired) and the CS-Only condiitons, rats were placed in the white compartment, facing away from the centre of the apparatus. For the CS-Only condition, the rats were placed in the left compartment. In the CS+CXT (paired and unpaired) and CS-Only conditions, the 10 s CS was presented when the rat had turned to face the centre of the apparatus, and the sliding was opened coincidentally with the offset of the CS. In the CXT-Only condition, the sliding door was opened approximately 10 s after the rat turned toward the opposite compartment. As in experiment 1, each rat received one test trial and no foot-shock was administered.

6. Results CO and OC groups did not differ on any of the analyses of experiment 2 and, as a result, for each condition, these groups were combined into a single control group. During pretraining, HPC and control groups displayed a strong preference for the black compartment of box 1 but spent equal amounts of time in the left and right compartments of box 2. As in experiment 1, there were no group differences on these measures within or across experimental conditions. The results of the CS+CXT (paired) condition did not differ in acquisition of the of the conditioned suppression response (see Table 3). When tested in the presence of the shock-associated CS and contextual background, both groups waited almost 200 s before entering the black compartment for the first time and, overall, spent the majority of their time in the white

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compartment. By comparison, there was no evidence of conditioned fear in the CS+CXT (unpaired) condition, where both groups displayed essentially the same preference for the black compartment as in pre-training. In the CXT-Only and CS-Only conditions, where rats were tested only in the presence of the context or CS respectively, HPC groups exhibited conditioned fear behaviour relative to control groups. There was a strong lesion effect in the CXT-Only condition as the HPC group exhibited longer response latencies and spent less time in the shock-associated black compartment than the control group. The group differences on latency (t(14)= 2.94, P B 0.01) and cumulative time spent in the black compartment (t(14) = 2.75, P B 0.02) were confirmed by t-test. In the CS-Only condition, there was a significant group effect on the response latency measure (t(12)= 2.24, PB0.05), indicating that the HPC group was slower to enter the right side of the box where the CS had been presented. The HPC group also displayed a tendency to spend less time in the right compartment during the test session, but this effect was not statistically significant, (t(12) =1.83, P \ 0.05). ANOVA was performed on the response latency and cumulative time scores in the CS-Only and CON-Only conditions to assess the differential effects of type of cue on the groups’ behaviour. This analysis revealed an overall lesion effect on both measures (P’s B 0.01). Although the differences between groups was consistently less in the CS-Only condition, neither analysis yielded a significant interaction.

Table 3 Mean response latencies (RL) and cumulative time (CT) spent in the shock-associated compartment of experiment 2 (a)

CS+CXT HPC

Control

Paired RL (s) S.D. CT (% of total) S.D.

192.4 27.6 25.7 9.0

118.3 26.8 45.7 7.7

Control

Unpaired 179.1 41.1 31.1 10.3

CS-only RL (s) S.D. CT (% of total) S.D.

HPC

45.8 12.6 75.5 9.4

49.5 10.1 78.9 8.2

CXT-only 92.0 15.5 53.4 8.0

171.3 444.8 22.3 8.7

109.9 38.6 36.3 11.5

(a) Mean RLs and CTs spent in shock-associated black compartment of box 1 during testing in CS+CXT conditions of experiment 2. (b) Mean RLs and CTs spent in shock-associated compartment during testing in CS-Only and CXT-Only conditins of experiment 2.

7. Discussion The results of experiment 2 are consistent with those of experiment 1 in showing that rats with HPC lesions form strong associations between salient contextual stimuli and shock-induced fear in a Pavlovian conditioning paradigm. The finding that the HPC group avoided the black compartment in CXT-Only testing when conditioned fear was measured by response suppression shows that the effect is not restricted to one type of response. Moreover, the ability of HPC groups in the CS+CXT (paired) and CXT-Only conditions to avoid the shock-associated area argues against a deficit in the inhibitory control of behaviour [4,8]. In one respect, the results of experiment 2 were not entirely consistent with those of experiment 1. In the previous experiment, the CS, presented in a neutral context, was more effective than contextual stimuli in evoking shock-avoidance behaviour in all groups. In experiment 2, this pattern was reversed as the groups generally had shorter response latencies and, in the CS-Only condition, spent more time in the right compartment (where the CS appeared) than in the black compartment in the CXT-Only condition. Moreover, the CS was less effective in differentiating the behaviour of the HPC and control groups, as group differences in the CS-Only condition emerged only on the response latency measure. The reason for this pattern is not entirely clear but task-related factors are probably important. In the procedure followed in this paradigm, rats learned to cross over to the other compartment during initial shaping. As a result, a discrete CS that was paired with footshock, may have evoked locomotion, creating a tendency in the animal to move to the other compartment. It is noteworthy that, notwithstanding this tendency, the HPC group still had longer latencies than controls, indicating a greater reluctance to enter the area where the CS had been presented. However, once in the other compartment, there was little incentive to leave since the CS had terminated and there were no contextual cues to signal conditioned fear. Under those conditions, it is not surprising that HPC and control groups were not highly motivated to avoid the right compartment.

8. Anatomical report Reconstructions of typical HPC and CO lesions are presented in Fig. 1. Damage produced by HPC lesions was similar to that described in previous reports involving identical surgical procedures [32,38]. Damage was restricted to the dorsal portion of the structure, with the area primarily affected lying between 3.0 and 5.0 mm anterior to the interaural plane. Invariably, damage to overlying

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Fig. 1. Diagrammatic representations of typical hippocampal (top) and prefrontal cortical (bottom) lesions.

cortex and corpus callosum accompanied the HPC lesions. Less frequently, there was partial destruction to cingulum, hippocampal commissure, fimbria, dorsal fornix, and, in a few cases, the lesion slightly invaded the dorsal thalamus. There was no evidence, in any of the groups, that site or extent of extra-hippocampal damage was related to performance. The CO lesions generally were smaller than the HPC lesions, but typically destroyed more cortex. CO lesions caused extensive damage to corpus callosum and frequently invaded the cingulate area. There was no difference in performance between CO rats with and without cingulate damage in any of the conditions.

9. General discussion A major purpose of the present study was to test the idea that HPC damage disrupts the normal response to stimulus elements in a complex environment. A classical fear conditioning paradigm was used in which a discrete CS (tone) was paired with a US (foot shock) in a distinctive context. During testing, the CS or the context was presented alone and their respective abilities to evoke the learned fear response was measured by the rat’s preference for a compartment that was not associated with shock. On the basis of previous work

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involving a similar paradigm [38] and the extensive literature on context and fear conditioning [1,2], it was predicted that control rats would combine the CS and CXT into a compound stimulus to which the fear response would become conditioned. Consequently, either stimulus alone should produce little fear behaviour. Rats with HPC lesions, because of their difficulties in forming new relationships between stimuli, would respond to the CS and CXT as segregated conditional stimuli rather than as an organized unit. The predictions were generally confirmed in two experiments that differed primarily in terms of the measure of conditioned fear. In experiment 1, fear was measured by the tendency to leave a shock-associated area and, in experiment 2, by response suppression in the presence of cues that signalled shock. In both experiments, groups that received cue-shock pairings were less fearful when tested in one of the shift conditions than corresponding groups in the CS+ CXT (paired) condition, where the same cue combinations were present during training and testing. However, the HPC groups generally found the separately presented cues to be more aversive than the control groups. This was especially apparent in the CXT-Only conditions of experiments 1 and 2, and the CS-Only condition of experiment 1. The differences between fear behaviour measures in these conditions and those of the CS+ CXT (paired) condition were much less in the HPC than in the control groups. A significant aspect of the present results is that, regardless of the response measure, CXT presented alone evoked strong fear behaviour in HPC rats. Several reports of impaired fear conditioning to context in rats with HPC lesions are based on studies in which response suppression was the measure of fear [15,24,39]. The present findings provide evidence that the dependent variable is not the critical factor in determining the effects of HPC damage on contextual conditioning. The results of experiment 2 also rule out the possibility that group differences in the CS-Only and CXT-Only conditions of experiment 1 were due to differences in initial strength of conditioning. In experiment 1, there was a possibility that ceiling effects masked differences between HPC and control groups in fear conditioning as measured in the CS + CXT (paired) condition (see Table 2). In the CS+ CXT (paired) condition of experiment 2, the groups did not differ on the response latency and cumulative time measures, and none of the scores approached ceiling levels (see Table 3). The CXT-Only test condition may be compared with other Pavlovian conditioning studies in which animals with HPC damage and controls received CS-US pairings in a distinctive context [7,25,38] and were subsequently tested in the same context but with the CS

altered or removed. The consistent finding in these studies, and the present one, was that normal animals displayed a response decrement following the change, while HPC animals continued to respond at the same rate. These studies, which involved conditioning of aversive and appetitive responses, show that when the discrete CS and CXT are both strongly associated with a US, animals with hippocampal lesions, in contrast to controls, form direct associations between the contextual cues and the US. In the absence of the CS, the CXT alone serve as an effective signal for the US and elicit conditioned behaviour in the lesioned animals. In the CS-Only condition, where the CS was presented in a totally different context, similar results were obtained. In experiment 1, rats with HPC lesions displayed a stronger fear response to the CS than controls by quickly vacating the compartment in which the CS was presented and avoiding it for most of the trial. The pattern was similar in experiment 2 (although, here, the group difference on the cumulative time measure did not reach statistical significance). By comparison, the control groups exhibited relatively little fear behaviour in response to the CS. The only other studies that tested this type of transfer behaviour in animals with HPC lesions were those of Penick and Solmon [22] and Honey and Good [11]. These investigators examined the effect of switching contexts on performance of conditioned responses in HPC-damaged animals. Consistent with the present results, they found that the CS continued to evoke the CR in the novel context in HPC groups, but not in control groups. The finding that contextual cues exert abnormally strong control over the behaviour of animals with HPC damage appears to be at variance with the view that HPC damage interferes fundamentally with the ability to use contextual information [7,10,15]. This conclusion is supported largely by evidence that animals with HPC damage are impaired on tests of conditional discrimination learning (CDL) in which combinations of discriminative stimuli and CXT are associated with different responses [7,9] but see [27]. Since HPC lesions do not usually affect performance on conditional learning tasks that require more straightforward associations between discriminative stimuli and specific responses [13,32], the observed deficits have been attributed to a failure to form contextual associations. In fact, CDL tasks, in which contextual stimuli are part of the discriminanda, have special features that explain their sensitivity to effects of HPC damage. Such tasks demand that animals form an association between discriminative stimuli and CXT, and relate each combination to a different response. The process of forming a unique representation of the stimulus relationships serves to differentiate each stimulus combination from

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others with conflicting associations, thereby reducing the potential for interference1. During testing, the animal is rewarded only for responding when the component stimuli are presented together; a response to either component in some other combination will not have the desired effect. In these tests there is no functional distinction between discriminative stimuli and CXT in that both are equal partners in the conditional relationship. The critical requirement is not that animals respond in a unique way to context, but that they organize key stimulus elements into unique representations that, in turn, enter into other relationships. Current theories differ in terms of their characterization of HPC function but there is general agreement that damage to the structure disrupts the ability to associate unrelated stimuli and form the types of unique representations that are necessary to perform CDL and similar tasks [6,17,28]. In an influential study, Kim and Fanselow [15] compared HPC and control rats on a test of memory for a fear response conditioned to a CS and CTX stimuli at variable intervals before surgery. When tested post-operatively in the same context, but without the CS, HPC groups exhibited the pattern of retrograde amnesia (RA) that is characteristic of HPC damage [31,40]. When the CS was presented in a different context, there was no evidence of RA as HPC and control groups displayed equally robust fear behaviour. This outcome appears to conflict with the present results which suggest that rats with HPC lesions tend to condition responses separately to CS and CXT. However, a crucial procedural difference is that, in the present study, different groups of rats were tested in the CS-Only and CXT-Only conditions. In Kim and Fanselow’s [15] experiment, rats in each training-test interval were administered both the CS-Only and CXT-Only tests, with the CXT-Only test always presented first. It is possible that initial testing in the original context primed the HPC group, thereby increasing the probability of the fear response in subsequent testing with either stimulus component. Indeed, Winocur and Black [33] showed that prior exposure to contextual cues associated with footshock can help HPC rats retrieve an apparently forgotten fear response when tested later in the presence of other shock-related cues. Another important procedural variation between studies of contextual fear conditioning relates to the nature of the CXT that are paired with foot-shock. In the present study and in others that have demonstrated significant fear conditioning to context in animals with HPC damage [7,23,29,38] CXT were highly distinctive and confined to the animal’s immediate environment. 1

Increased susceptibility to interference is a widely reported effect of HPC damage [12,30] that has been attributed recently to a fundamental deficit in associative learning [26].

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In some experiments, where HPC lesions impaired contextual conditioning, an array of local stimuli combined to form a complex environmental background [24,39]. As Young et al. [39] point out, for contextual fear conditioning to occur under such circumstances, the animals must form an integrated representation of environmental stimuli that constitute the learning context. The process of integrating spatially-distributed environmental stimuli in this way may help animals form a superordinate context that helps to define an event. In view of the large body of evidence that HPC lesions interfere with this type of processing [3,19,22], it follows that animals with HPC lesions would be impaired in contextual conditioning tasks in which it was necessary to create an integrated representation of context (see [20] for a similar account). By comparison, no such integration was required in the present task where a highly salient and non-differentiated black box served as the single contextual cue. Other findings show that animals with HPC damage have no difficulty associating CXT with learned behaviour. For example, Good and Honey ([7], experiment 3) trained HPC and control groups of rats to associate one context with food delivery and another context with no food. They tested the ability to discriminate between contexts by placing the rats in either context and measuring approach behaviour for food. There was no difference between HPC and control groups as both responded at significantly higher rates in the context associated with food. Similar results were obtained by Skinner et al., [27] in an investigation of the effects of HPC damage on conditioning a tasteaversion response to context. An important point is that these examples parallel studies in which HPC lesions failed to disrupt performance on tasks where virtually the same associations were made with discrete CSs or discriminative stimuli rather than contextual cues [5,18]. The involvement of the HPC in learning and memory depends on the type of associations that must be made between stimuli, whether they appear as CSs or background stimuli. Tasks on which HPC animals perform normally are those in which they can form direct associations between salient environmental stimuli and responses that reliably predict reward. Thus, HPC damage does not affect most forms of classical conditioning, simple operant conditioning, and discrimination problems in which stimulus-response associations are readily established [3,8,22]. In the present study, rats with HPC lesions had no difficulty acquiring the fear response, which was predicted with high probability by the discrete CS and by contextual cues. In previous work, using instrumental conditioning procedures [34,37], we demonstrated context-specific discrimination learning in rats with HPC lesions, such that when the context was drastically changed, HPC

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groups, but not controls, responded less accurately to the same discriminanda. The groups did not differ when discrimination training and testing were conducted in the same context. This finding contrasts with the present results and those of other studies (eg. [11,23]) that used classical conditioning paradigms and found that a change in context produced response decrements in control but not HPC groups. In fact, these results, based as they are on entirely different paradigms, should not be seen as contradictory. Context influences behaviour in many ways and, under certain conditions can act as an occasion-setter or as a retrieval cue that signals when a particular stimulus-relationship is in effect [2]. In the earlier papers, we argued that, because of a lesioned-induced sensitivity to distinctive environmental cues, rats with HPC lesions formed an association between the discrimination tasks and the unique contexts in which they were presented. When the contexts were altered at test, HPC rats were slow to recognize the original task and went through a period of confused relearning. On the other hand, the familiar discriminanda and possibly other cues were sufficient for control rats to recognize the task, and they proceeded to demonstrate relatively good savings. Taken together, the results indicate that, while HPC lesions alter the animal’s response to context, the expression of that change varies with the task and relates to the various ways that contextual cues are used. The disruptive effects of HPC lesions on contextual information processing are seen as contributing to the memory deficits that are characteristic of damage to this structure. It is instructive that the types of contextrelated changes observed in HPC animals occur in analogous form in amnesic patients with known or suspected damage to HPC and related structures (eg. [35,36]). Unable to form the associations that define an event within its context (the term, ‘context’ is used here in the broad sense to include relevant spatial and temporal attributes), HPC-damaged animals or humans cannot meaningfully represent an experience and, as a result, accurately recall it. HPC damage forces individuals to resort to relatively inefficient learning strategies that permit only direct associations between distinctive stimuli and unconditioned stimuli or reinforced responses. These associations can support context-free forms of habit or procedural learning, but not explicit or declarative memory that is based on the recall of context-specific information. The present results indicate that rats with HPC lesions do not differentiate between distinctive contextual cues and other discrete stimuli that are reliably associated with reward. As a result, they are unable to use either form of stimulus to create relationships with other stimuli [10] or to engage in the type of relational or associative learning described respectively by Eichen-

baum and colleagues (eg. [3]) and Sutherland and Rudy [28]. The results of this study are generally consistent with those of other studies that point to impaired contextual processing following HPC damage. If there is a point of departure, it is with respect to whether the deficit is restricted to contextual information and whether HPC lesions abolish responding to contextual cues. The view favoured here is that the fundamental deficit is related to associative processes, is not specific to contextual information, and is characterized by an alteration rather than elimination of the response to context.

Acknowledgements This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. The author is grateful to M. Moscovitch, A.R. McIntosh, and N. Meiran for having commented on an earlier version of the paper.

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