Successful overshadowing and blocking in hippocampectomized rats

Behavioural Brain Research, 12 (1984) 39-53

39

Elsevicr BBR 00340

SUCCESSFUL OVERSHADOWING AND BLOCKING IN H I P P O C A M P E C T O M I Z E D RATS

P. G A R R U D 1, J.N.P. RAWLINS 2, N.J. M A C K I N T O S H 3, G. G O O D A L L 4, M.M. COTTON 5 and J. F E L D O N 6

~Department of Psychology, St Andrews University, St Andrews, Fife, K Y16 9JU, 2Department of Experimental Psychology, University of O~xford, South Parks Road, Oxford OXI 3UD, 3psychological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EB (U.K.), 4Laboratoire d'Ethnologie et Sociobiologie, Univers#O de Paris XIII, Villetaneus 93430 (France), ~Department q[ Psychology, University of Newcastle, Newcastle, New South Wales (Australia) and 6Department of Psychology, Tel A viv University, Ramat A viv (Israel) (Received January l lth, 1984) (Accepted March 1st, 1984)

Key words: hippocampus - conditioned suppression - overshadowing - blocking - rat

Overshadowing (Experiment 13 and blocking (Experiment 2) were investigated using a conditioned suppression paradigm in rats. Neither hippocampectomy nor cortical control lesions affected the extent to which a salient stimulus overshadowed a less salient one. Nor did the lesions affect the extent to which a stimulus that was highly correlated with shock overshadow a stimulus that was less well correlated with shock. Finally, the lesions did not alter the extent to which a previously reinforced stimulus blocked conditioning to another stimulus when both were presented as a reinforced compound stimulus. It is thus possible for hippocampectomized rats to show apparently normal overshadowing and blocking, at least under some testing conditions.

INTRODUCTION

Among the many functions attributed to the hippocampus on the basis of information derived from lesion studies has been that of tuning out irrelevant or uninformative stimuli. This suggestion was first made by Douglas 5'6, and has since been further elaborated 16"17"29"3°. These more recent accounts introduced an important distinction between those stimuli which signal nothing, or no outcome not already predicted by another more informative stimulus, and those stimuli which signal the absence of reinforcement a highly informative event if there are other stimuli present which predict that reinforcement will occur. The importance of this distinction was made clear in a series of experiments on nictitating membrane conditioning in the rabbit. Ablation of dorsal hippocampus had little effect on the acquisition of conditioned responding to a light which signalled the occurrence of a brief shock to the 0166-4328/84/$03.00 © 1984 Elsevier Science Publishers B.V.

cheek, and equally failed to disrupt the discrimination formed, when trials on which a tone was added to the light and the compound was not followed by shock were randomly interspersed among trials on which the light alone was presented followed by s h o c k 29. Inhibitory conditioning to the tone, which signalled the absence of the shock otherwise predicted by the light, proceeded quite normally in rabbits with hippocampal lesions. The lesions did, however, have pronounced effects in two other experiments. In control animals, unreinforced pre-exposure to a tone subsequently used as a conditioned stimulus (CS) paired with shock, significantly interfered with the course of conditioning to the tone, a phenomenon usually, if rather misleadingly, termed 'latent inhibition'1°. Lesions in the dorsal hippocampus abolished this effect: lesioned rabbits conditioned to the tone at the same rate regardless of their previous exposure to it 3°. A second experiment 29 established that the lesion greatly attenuated the blocking effect originally reported by Kamin 9. In control ani-

40 mals, little or no conditioning occurred to a light paired with shock if it was always presented in conjunction with a tone that had previously been conditioned with the same shock. Prior conditioning to the tone blocked conditioning to the light, but this blocking effect was abolished by lesions in dorsal hippocampus: lesioned animals showed similar levels of conditioning to the light whether or not they had previously been conditioned to the tone. On the basis of these results, Moore and Stickney developed a relatively formal theory of hippocampal function ~7. A number of writers have interpreted latent inhibition as a failure to process or pay attention to an uninformative or redundant stimulus; its associability with reinforcement is said to decline as a consequence of non-reinforced pre-exposure (cf. refs. 11, 20, 31). Although blocking has often been interpreted in different ways (e.g. ref. 23), it has been argued that little conditioning accrues to the added CS in a blocking experiment because it is followed by an event already predicted by the previously trained CS. By establishing that hippocampal lesions have similar effects on latent inhibition and blocking, the data encourage the similar analyses provided by these theories ~1,2o,31 for both phenomena. The hippocampus is involved in tuning out redundant or uninformative stimuli. This may not be a complete account of hippocampal function in rabbits, for there is reason to believe that redundancy may not be the only feature of a stimulus determining whether it will be responded to in a similar manner by hippocampally damaged and normal animals (cf. refs. 28 and 2, investigating repeated extinction and reversal of a discriminated eyeblink response, respectively). But even data not at first sight providing strong support for this analysis may be consistent with it. There is evidence that hippocampally damaged rabbits may be impaired in trace conditioning of the nictitating membrane response 33. This may be because they are unable to tune out the contextual stimuli present during the trace interval and thus condition too strongly to such stimuli at the expense of the nominal CS. Further support for Solomon, and Moore and Stickney's analysis has been provided by a num-

ber of studies of conditioned suppression by Rickert and his colleagues, who have found a similar attenuation of blocking in hippocampally damaged rats 24"25. They have also shown that hippocampal lesions appear to interfere with overshadowing, a phenomenon usually thought to be closetv related to blocking 26 (see also ref. 27 L In Rickerl et al.'s experiment, modelled after an earlier study '2. rats received a series of conditioning trials to a compound CS consisting of a light and one or other of two tones. For one group, a random 50" of trials were reinforced, regardless of which tone was presented on each trial, and subsequent test trials revealed good evidence of conditioning to the light. A second group also received reinforcement on only 50° o of trialsL but in their case the outcome of each trial was perfectly predicted by the tone occurring on that trial: one tone ,sas always present on reinforced trials, the other only on unreinforced trials. Although, just as in the first group, the light was followed by reinforcement on 50~,o of trials, it was always presented in conjunction with a more informative stimulus. and animals in this second group showed little or no evidence of conditioning to the light alone (when tested) after the compound conditioning trials. As in the blocking experiments, this failure of conditioning to an uninformative stimulus was abolished by lesions in the hippocampus. Unlike control animals, lesioned rats showed just as much suppression to the light when it was accompanied by the more informative tones as they did when the two tones were uncorrelated with the outcome of each trial. The pattern of results observed in these experiments simultaneously supports the hypothesis that overshadowing, blocking and latent inhibition {but not conditioned inhibition) may be due to some common process, and suggests that one of the functions of the hippocampus is to mediate this process of tuning out or ignoring uninformative stimuli. The experiments reported below were designed to confirm and extend this analysis. One way of elucidating the nature of the process in question would be to see what other, more or tess similar, phenomena were affected by hippocampal lesions. For example, the overshadowing reported bv Wagner etat. ;~ and by Rickcrt

41 et al. ~4"25 is not the only form that has been reported. Ever since some studies of Pavlov 19, it has been known that conditioning to one element of a compound CS is adversely affected not only by the presence of another element better correlated with the outcome of each trial, but also by an increase in the intensity or salience of the other element (e.g. ref. 12). There is some question whether this form of overshadowing is due to the same process as that responsible for the failure of conditioning to a CS worse correlated with reinforcement than another ~3,8. One way of answering this question is to see whether hippocampal lesions have similar effects when the overshadowing stimulus is better correlated with reinforcement than the overshadowed stimulus and when it is simply more salient. EXPERIMENT I Experiment I was designed to permit simultaneous assessment of overshadowing of a visual stimulus by auditory stimuli better correlated with reinforcement and by auditory stimuli no better correlated than the light but whose intensity was significantly increased. In a Control condition animals were conditioned to a tone-light and to a clicker-light compound, both compound stimuli being reinforced on a random 50~o of trials. In a Salient condition, animals received exactly the same sequence of trials and reinforcement but the intensity of the two auditory stimuli was radically increased. In the Correlated condition, the clicker-light compound was always, and the tone-light compound never, reinforced, but since there were equal numbers of both kinds of trial, the schedule of reinforcement associated with the light was the same as in the control group. Test trials to the light alone were given at various stages of conditioning. Before the start of the experiment, animals were assigned to one of 3 groups for surgery: Hippocampal, Cortical control and Sham operated. There were thus 9 groups of rats.

Me~od Subjects and apparatus The subjects were 68 naive male Wistar rats from the colony maintained in the Department of Experimental Psychology, Oxford University. Twenty-six were allocated to the hippocampal lesion group, 24 to the cortical control lesion group, and 18 underwent sham operations. They were between 70 and 90 days old at the time of surgery. Four operant conditioning chambers were used (see ref. 4 for further details). They were equipped with a single lever, a recessed food magazine, a loudspeaker and a 240 V 30 W striplight mounted above the white Perspex ceiling. Each box was enclosed in a sound- and light-attenuating cubicle. The rewards used were Campden Instruments 45-mg food pellets. Scrambled shock could be applied to the grid floor from Grason Stadler E1064GS shock generators; all shocks were of 0.6 mA intensity for 0.5 s. The 3 CSs employed were as follows: a 1 kHz tone and a 20 Hz clicker from a Campden Instruments audiogenerator, with intensities of 64 and 70 dB (re 0.002 dynes/cm2), respectively, measured next to the lever; and a light produced by 60 V across the striplight. All CSs were 30 s long. White noise was played through the speakers except when the auditory CSs were presented.

Surgery and histology Operations were carried out under chlornembutal (containing 9.7 mg/ml sodium pentobarbitone and 42 mg/ml chloral hydrate) anaesthesia (3 ml/kg). All subjects were placed in a specially designed head-holder 22 which allowed the head to be fixed in any desired position. An opening was drilled in the skull overlying the dorsal hippocampus, using a dental drill. In the Hippocampal group, the dura was sectioned, and the necessary neocortex removed to allow the hippocampus to be aspirated. All aspirations were performed under visual control, using a Zeiss OPMi I operating microscope. The Cortical group had an equivalent area ofneocortex and corpus caUosum aspirated, exposing the alveus of the dorsal hippocampus. Both groups had the wound packed with

42 Sterispon absorbable gelatine foam soaked in physiological saline. The Sham operated group had a similar opening burred in the skull, but the dura was left intact: Sterispon was laid on the dura. Sulphanilamide powder was applied and the scalp sutured. Benzathine penicillin (Penidural) was injected (0.1 ml i.m.) into each hind leg. At the conclusion of the behavioural experiment, the rats in the cortical control and hippocampal lesion groups were given an overdose of sodium pentobarbitone, and perfused intracardially with physiological saline followed by 10~,',, formol saline. The brains were blocked, and embedded in celloidin. They were sectioned at 50/~m, with every 5th section being saved, and stained with cresyl violet. They were examined microscopically for direct damage and for gliosis. and the damage assessed at each of 3 planes of sectioning (see Fig. 1).

Procedure Three weeks after surgery, the rats were gradually introduced to a food-deprivation regime which maintained their bodyweight at 85 ~o of its free-feeding value. All rats were then given one session of magazine training with the levers removed from the boxes, and in which 45 mg pellets were delivered at varying intervals averaging 30 s. This was followed by one (or two in the case of rats who had to be shaped to lever press) session of continuously rewarded lever pressing: these sessions lasted 30 rain. They then received one session of variable interval 30 s lever press training, followed by t6 sessions of variable interva160 s training, with each session lasting 40 min. This schedule remained in effect for the remainder of the experiment. Before the start of conditioning, animals were assigned to one of 3 behavioural conditions: Correlated, Salient and Control. Within each lesion group, animals were divided into trios on the basis of their rate of lever pressing in the final 3 sessions of baseline training, and one animal from each trio was assigned to each condition. On Day 1, the day before conditioning started, animals received a single exposure to each of the following 3 stimuli: a tone-light compound, a clicker-light

compound, and a light. In the Salmnt condition the intensity of the tone and clicker, both on this day and for the remainder of the experiment, was set 26 dB higher than that specified for the other two conditions. Throughout conditioning there were 4 compound trials each day. two to the tone-tight compound, and two to rhc clicker-light compound. In the correlated condition, the cli~ cker-light compound was always, and the tone-light compound never, followed by shock. In the Salient and Control conditions, one t o n e light and one clicker-light trial each day were followed by shock. The order of trials and shocks was varied quasi-randomly from day to day, as was the interval between trials, although the minimum interval was always 5 mm in addition to the 4 compound trials each da), there were 3 days (6, 13 and 20) on which all animals also received two test trials to the light alone. The first light trial was never followed by shock, the second always was. On Day 22. some animals received one non-reinfbrced test trial to the tone and one to the cticke~ The experiment was run m two replications with only very minor differences between the two: only the animals in the second replication received the final test trials to the tone and clicker alone. There were unequal numbers of animals in each replication, condition and lesion group. These numbers are shown in Table I.

Results Histology All the 24Cortical control subjects had sustained bilateral neocortical damage exposing the alveus of the hippocampus (see Fig, 1). One rat was excluded from this group because the hippocampus was extremely distorted unilaterally and one rat was excluded because the cortical damage, though bilateral, was very restricted in extent. Of the remaining rats~ 3 bad restricted superficial alvear damage unilaterally; in the other subjects there were no discernible lesions in the hippocampat formauon. The cingulum bundle was undamaged in 14 subjects: unilaterally damaged. though never totally sectioned, in 6 subjects: and incompletely bilaterally damaged in 3.

43 TABLE I Numbers of subjects in each group Experiment 1 Condition

Correlated Salient Control

Lesion Hippocampal

Cortical

Sham

1st

2nd

1st

2nd

1st

2nd

4 4 4

5 5 4

4 4 4

4 4 4

4 4 4

2 2 2

Experiment 1I Condition

Blocking Control

Lesion Hippocampal

Cortical

Sham

10 9

9 9

9 8

CC

Fifteen subjects had sustained cortical lesions at the intermediate plane; and 5 subjects had lesions at the posterior plane, 3 of them bilaterally. All the 26 rats in the Hippocampal lesion group had sustained extensive bilateral hippocampal damage, and bilateral neocortical damage, (see Fig. 1). One rat had clear unilateral damage extending into the dorsal thalamus, and was excluded from the study. In the remaining subjects the thalamus had not been invaded, though in 3 there were restricted areas of gliosis unilaterally in the dorsal thalamus; these rats were retained in the study. The cingulum bundle was undamaged in 14 subjects; unilaterally damaged, though not completely sectioned, in 5 subjects; and bilaterally damaged in 6. In no case was there a complete bilateral transection. Six rats had unilateral entorhinal cortical damage. All the rats had bilateral hippocampal damage at the anterior plane of assessment, but only 12 had bilateral cortical dam-

HC-s

HC-L

Fig. 1. Photographs of coronal sections of brains stained with cresyl violet. The photographs illustrate the extent of damage at anterior (top), intermediate, and posterior (bottom) planes of assessment, in a rat with an approximately median cortical lesion (CC); in the rat with the smallest hippocampal damage to be retained in the study (HC-S); and in a rat with one of the largest hippocampal lesions (HC-L). "

44 age, with 3 having no cortical damage at all. At the intermediate plane, 6 rats had total bilateral destruction of the hippocampus, and the remaining 19 had bilateral damage extending well past the hippocampal flexure, and usually sparing only the most temporal pole of the hippocampus. Seventeen subjects had no neocortical damage at the most posterior plane of assessment, but all the subjects had hippocampal damage at this plane. the damage being bilateral in 17 of them. Thus, in general, the neocortical lesions in the two groups were comparable in size and in location. The hippocampal lesions were extensive and bilateral, usually producing total ablation of the structure at some levels of sectioning, particularly towards the 'adseptal '1 pole. and almost always extending further temporally than the hippocampal flexure. In a quarter of the animals, the damage to the ventral portion of the hippocampus was also complete at some planes of sectioning, with only the most temporal pole remaining intact.

Behaviour Pre-CS rates. Performance on conditioning and test trials was measured by calculating suppression ratios, of the form a/(a + b), where a = the number of responses during a CS and b = the number of responses during the 30 s preceding the CS. In order to provide an estimate of the overall rate of responding at various stages of the experiment, we also analyzed pre-CS rates on the 7 trials on which the light was presented alone (one pre-test, and 3 test days with two test trials per day). Analysis of variance revealed significant effects of trials (F6, 282 = 16.88, P < 0.001), reflecting an overall increase in pre-CS rate from 23.2 responses per minute on pre-test to 42.1 responses per minute on the final test trial, and of lesion (F2, 47 = 3.63, P < 0.05) with response rates of 40.0, 24.3 and 31.8 for Hippocampal, Cortical and Sham operated animals, respectively. The interaction between lesion and trials was not significant (F 12, 282 = 1.49, P > 0.10). There was also a significant effect of replication (F 1. 47 = 5.49, P < 0.05) and interaction between replication and trials (F6, 282 = 4.46, P < 0.001): animals in the first replication responded more slowly than those in the second (26.5 vs 37.6

responses per minute), and the interaction with days reflected the fact that this difference was more marked on the pre-test and final pair of test trials than on the intervening trials. No other effect (including no effect of condition or interaction between condition and lesion) was significant (maximum F = 1.49). CS pre-test. On the 3 pre-test trials, overall suppression ratios were 0.23 to the tone-light, 0.21 to the clicker-light and 0.31 to the light alone. Analysis of the tone-light and clicker-light suppression ratios showed a significant interaction between Replication. Lesion and CS type (F2. 47 = 3.49, P < 0.05); the Sham animals in the second replication suppressed more to the clicker-light compound than to the tone-light (0.05 vs 0.29). This difference in suppression was not apparent on the following da~ (Day 1 of conditioning: suppression ratios: 0.16 and 0.t6). Light pre-test. The extent of the unconditioned suppression to the light differed in the two replications (0.38 in Replication 1 vs 0.23 in Replication 2: F 1.47 = 10.38. P < 0.01 ): moreover the extent of the suppression differed in the rats allocated to the different behavioural conditions (Correlated group, Replication 1 = 0.33, Replication 2 = 0.37: Control group, Replication 1 = 0.41, Replication 2 = 0.17; Salient group, Replication 1 = 0. 40, Replication 2 = 0.16: F 2, 47 3.91. P < 0.05). Compound conditioning. The course of discriminative conditioning to the clicker-light and tone -light compounds in the Correlated condition over the 20 days of conditioning is shown m Fig. 2. Suppression to the reinforced clicker-tight compound was acquired in all 3 lesion groups, and all 3 eventually lost suppression to the nonreinforced tone-light compound. An overall analysis of variance was undertaken including the Salient and Control conditions. There was a significant main effect of days (F 19, 893 = 12.27. P < 0.001); and significant interactions between Condition and CS type (clicker-light or tone -light) F2. 47 = 22.02. P < 0.001); and between these two factors and days (F38, 893 = 3.60. P < 0.001). These interactions reflect the fact that only in the Correlated condition, as shown in Fig. 2. did suppression to the two CSs diverge

45

0.5-

• 0

Shom

TONE- LIGHT

A

Hipp0campa[

Corficol

CLICKER - LIGHT

-.0

0.1+-

, ,

lb.

."k

o

~

,q /",

A

0.3-

I "A~

II

,, ~

~'l/ "/","k"k 7X~" ," "¢ , ,'.',"\'~ ,~.% ,r-"

0 I/I L/I

"

I

Ii

~-L. \ ~-.°" M , I "~ ~\~,

/

~e 4

.---"" ,--

, \

~ ./

.J

,*~ ' ,'

,/

"'

// ~ ".,.~,.~

i

II

\

~__4~'.o.

/

/

,~' ,,o"

,' 9 " ~ " "

~" "-o-"

/

~0.2I./)

~ 0.1

0

N

--

I

l(P)

.

.

.

.

I

.

.

.

.

.

.

6

I

.

.

.

.

'

'

13

I

20

Day Fig. 2. Experiment •:suppressi•nrati•st•t•ne-•ightandc•icker-•ightc•mp•undsduringthec•urse•fdiscriminativec•nditi•ning in the correlated condition; P is CS pre-exposure day.

over the course of conditioning. Separate analysis on the data from reinforced clicker-light trials revealed a significant effect of days (F19, 1064 = 10.07, P < 0.00l), but no effect of conditions or lesion and no interaction between any of these factors (maximum F = 1.07); however, on tone-light trials there were significant effects of days (F 19, 1064 = 5.82, P < 0.001) and conditions x days (F38, 1 0 6 4 = 3 . 7 3 , P < 0 . 0 0 1 ) . There was no effect of lesion and no interaction involving lesion (maximum F = 1.16). Test trials. The data from the test trials to the light are shown in Table II. The analysis of variance on these data included the results from the pre-test trial to the light. The analysis found significant main effects of days (F4, 188 = 11.55, P < 0.001) and of conditions (F2, 47 = 7.06, P < 0.01), and a significant interaction between the two (F 8, 188 = 4.04, P < 0.001). Neither the

TABLE II M e a n suppression ratios on test trials to light alone in E x p e r i m e n t 1

Lesion

Hippocampal

Cortical

Sham

Condition

D a y s (pre-exposure) 1

6

13

20

Correlated Control Salient

0.33 0.21 0.27

0.12 0.12 0.27

0.35 0.25 0.33

0.41 0.27 0.28

Correlated Control Salient

0.33 0.38 0.32

0.18 0.18 0.26

0.28 0.18 0.20

0.40 0.20 0.32

Correlated Control Salient

0.39 0.27 0.25

0.16 0.11 0.30

0.41 0.16 0.23

0.41 0.18 0.32

46 main effect of lesion nor any interaction involving lesion was significant: the only interaction approaching conventional levels of significance was that between lesion, conditions and days (F16, 188= 1.67, 0 . 1 0 > P > 0 . 0 5 ) . From inspection of the data this seems to reflect some loss of suppression to the light on the last test day by Hippocampal animals in the Control condition. The nature of the interaction between days and conditions can be seen in Table II, and was tested by N e w m a n - K e u l s tests. There was no difference between conditions in suppression to the light on the pre-test trial; on the first test day, however, there was significantly less suppression to the light in the Salient than in the Control condition, and on the second and third test days there was significantly less suppression in the Correlated than in the Control condition (P < 0.01 in all cases). The presence of these effects in each lesion group taken alone was assessed by separate analyses of variance on the data from the first and third test days. The interaction between days and conditions was significant in each group: for Hippocampals, F 2 , 19 = 5.55, P < 0.02; for Corticals F 2 , 1 6 = 5.36, P < 0 . 0 2 ; for Shams, F 2 , 12 = 7.37, P < 0.01. Although there were occasional significant main effects of replication in some of these analyses, and 3 interactions between replication and days, there was no interaction between replication and either conditions or lesion. In the second replication only, animals were finally tested for suppression to the clicker and to the tone alone. There were significant differences between the 3 conditions in suppression to the tone (F2, 22 = 4.29, P < 0.05); the mean suppression ratio in the Correlated condition was 0.48; in the Salient condition 0.38; and in the Control condition 0.22; but there were no differences in suppression to the clicker (F < 1). In neither case was there any effect or interaction due to lesion (maximum F = 1.18). Discussion The results may thus be succinctly summarized: the presence of intense auditory stimuli or of less intense stimuli better correlated with rein-

forcement resulted in significam overshadowing of conditioning to the light. In the Correlated condition such overshadowing developed slowly, as animals learned the discriminauon between reinforced clicker-light and unreintorced tone-light trials. In the Salient condition, overshadowing was present at the first time of testing to the light alone, after 5 days of conditioning, but seemed to decline with continued training. With the possible exception of this fin al. tentative suggestion, these results are very much what previous data and existing theory would lead one to expect. What was tess expected was that all these effects were as readily observed in animals with extensive hippocampal damage as in controls. E X P E R I M E N T 1I

The results of Experiment 1. although surprising, seem entirely clear. Rather than attempting a further replication of these results, we turned to the phenomenon of blocking to see whether we could obtain evidence that hippocampal lesions would reduce the extent to which prior conditioning to one element of a compound CS would block conditioning to the other element. Me~od Subjects and apparatus The subjects were 54 naive male albino rats, from the same stock as those used in Experiment I, which were allocated to 3 groups: Hippocampal, Cortical and Sham, with 19. 18 and 17 rats in each, respectively. The apparatus was the same as before; the 3 CSs. tone, clicker and light were those used for Correlated and Control conditions in Experiment I, and the US was the same 0.5 s. 0.6 mA shock Procedure Details of surgery, recovery, deprivation and preliminary training were the same as in Experiment I, the only difference being that the amount of training in the VI 60 schedule before conditioning started was reduced to 8 days. All animals were given one pre-exposure to each of the 3

47 stimuli used as CSs, tone, clicker and light, in a single session. For the first stage of conditioning, the animals were divided into two main groups, Blocking and Control, the former being conditioned to the tone (CS L), the latter to the clicker. Both groups received two reinforced conditioning trials in each session, separated by at least 5 min. Stage 1 lasted 9 days, for a total of 18 trials. In Stage 2, both groups received two reinforced tone-light (CSt + CS2) trials on each day for 4 days. After this compound training, all animals received two test sessions; in the first there were two non-reinforced presentations of the light (CS2); in the second session there were two further non-reinforced light trials, followed by a single non-reinforced trial to the tone. The number of animals in each lesion group and behavioural condition are shown in Table I. The histological procedures were as in Experiment I.

Results Histology All the cortical control subjects except one had received bilateral neocortical damage, exposing the alveus of the hippocampus. The one exceptional animal was excluded from the study. A further 7 rats were excluded from the study because they had sustained clear penetration lesions into the hippocampal formation. In two subjects the main cortical damage was anterior to the anterior plane of assessment and did not extend as far posterior as the intermediate plane. In the remainder there was clear, bilateral neocortical damage at the anterior plane of assessment, and neocortical damage at the intermediate plane, which was bilateral in all but 3 rats. The extent of the lesions, and encroachment upon the cingulum bundle was similar to that reported in Experiment I. The hippocampal lesions were also very similar to those described earlier: all the rats had sustained extensive bilateral hippocampal damage, and bilateral neocortical damage. One rat died during lever press training. One rat was excluded from the study because an infection had caused extensive extra-hippocampal damage. Two rats were

excluded because they had unilateral lesions extending into the thalamus. In the 15 rats retained in the study, there was unilateral entorhinal cortical damage in 4; the other 11 subjects had sustained no entorhinal damage. Hippocampal damage at the anterior plane was total in 6 subjects at the anterior plane of assessment, but was total at the intermediate plane in only one. There was hippocampal damage at the posterior plane of assessment in all the rats, the damage being bilateral in 8 of them. The extent and nature of the hippocampal and extra-hippocampal damage was very similar to that described in Experiment I.

Behaviour Pre-CS rates. As in Experiment I, the 3 lesion groups showed significant differences in their PreCS rates of lever pressing (Hippocampal, 37.4; Cortical, 27.6; Sham, 22.5 responses per minute; overall analysis F2, 35 = 3.71, P < 0.05). The CS data were therefore transformed into suppression ratios for analysis, as before. CS pretest. The 3 different stimuli produced different degrees of unconditioned suppression (Tone, 0.29; Clicker, 0.41; Light, 0.17: F2, 70 = 23.9, P < 0.001). The extent of the suppression was not significantly changed by the lesions, though there was a tendency for the Hippocampal group to show less, and the Cortical group more, suppression than the Sham operated controls (Means: 0.33; 0.24; 0.30, respectively: F2, 35 = 3.06, 0.10 > P > 0.05). Stage 1 ."single-stimulus conditioning. The results for Stages 1 and 2 are shown in Fig. 3. All the subjects showed clear conditioned suppression (see Fig. 3), which developed at much the same rate and to the same extent in all groups. (Days: F 8,280 -- 96.1;P < 0.001). There were no significant interactions with the Lesions or Conditions (maximum F = 1.57). Stage 2: compound conditioning. As expected, the control group (which had been conditioned using a clicker in the previous stage) differed from the blocking group (which had been conditioned using a tone) in their degree of suppression to the tone-light compound (F 7, 35 = 9.00, P < 0.01). However, as can be seen in Fig. 3, during

48

Stage 1 •

Stage 2

Test

Sham

0 Corhccat /x Happocompo[

0.5-

/

Confro, (El B[ockmg (8)

O.t,-

?

!

o

/

"It

/

c= 0.31 tO of

,%'0 O

C~

~0.2t~

il

A

0.1-

0

..b-~

i

I

2

I

I

t,

I

I

I

I

6

8

i

10

"~

12

L1

--

l

L2

T

Days Fig. 3. Experiment I1: Suppression ratios to the tone or clicker in Stage 1, tone-light compound in Stage 2. and on the first two test trials to the light (LI and L2) and a single test trial to the tone (T).

compound conditioning the Control group showed a steady increase in suppression, while the Blocking group did not, so that by the end of this stage the two groups showed equivalent suppression. There was a significant effect of days, F 2 , 105 = 7.04, P < 0 . 0 0 t ; and an interaction between Condition × Days, F 3 . 105 = 6.63. P < 0.001. There were no significant effects including Lesion as a factor (maximum F = 1.79). Test phase. Fig. 3 shows suppression ratios to the light on the first two test trials only, since by Trials 3 and 4, substantial extinction of suppression was evident. Statistical analysis was also restricted to Trials 1 and 2. The Blocking group showed significantly less suppression to the light than the Control group, (F1, 36 = 24.09, P < 0.001). This outcome was not affected by the lesions: there were no significant effects including

Lesion as a factor (maximum F = 1.77). Independent analyses of variance, conducted within Lesion groups, confirmed that there was significant evidence of blocking in each group: for Hippocampals, F 1, 13 = 8.91; for Controls, F 1 , 10 = 14.37; for Shams, F 1, 15 = 6.69; P < 0.02 in all cases. There were no significant interactions including any other factor, though there was a non-significant tendency for all the animals to show less suppression on the second non-reinforced trial. (F 1. 36 = 3.72. 0.10 > P > 0.05). The results of the final test trial to the tone are also shown in Fig. 3. The Control animals showed significantly less suppression than the Blocking group (F 1, 35 = 16.87. P < 0.001). Lesion was not significant as a factor and entered into no interaction (maximum F = 1.36). The results of this experiment are, if anything,

49 even clearer than those of Experiment I. There was no difficulty in obtaining a significant blocking effect, but not the slightest suggestion that blocking was affected by damage to the hippocampus. The 3 lesion groups behaved similarly at all stages of the experiment. GENERAL DISCUSSION

The discrepancies between the results reported here and those reported earlier 24-26 might be attributable to either of two sources: differences in physiological manipulations, or differences in behavioural treatment. The lesions in the hippocampally damaged rats used in this study were very extensive, including all the subfields of Ammon's Horn, and the dentate gyrus. There is no generally accepted dividing line separating the 'dorsal' hippocampus from the 'ventral' hippocampus, and relatively little cytoarchitectonic or hodological basis for making such a division; it is therefore difficult to assess critically the possibility that different behavioural consequences might result from lesions preferentially implicating the dorsal or ventral parts of the hippocampal formation. However, it is clear that, partly due to damage to fibres en passage, damage near the adseptal pole of the hippocampus might have physiological consequences rather different from damage near the temporal pole. The former, if including the fimbria and the posterior portion of the fornix, would effectively separate the septurn from the hippocampal formation, resulting in a complete loss of theta activity in the remaining portions of the hippocampal formation, and in the entorhinal cortex 21A5 and a loss of the hippocamposeptal projection from CA31,14. However, hippocampal-entorhinal relations would remain largely unchanged, since the projection to the entorhinal area from CA3 is derived from the temporal third of the hippocampus7. Damage near the temporal pole would damage this pathway, but leave septohippocampal relations largely intact. Our failure to affect blocking or overshadowing by making hippocampal lesions cannot convincingly be attributed to inadequate hippocampal damage; aspiration lesions of the type used here

are generally notably large, and include all the hippocampal subfields and the dentate gyrus, and often invade the retrohippocampal areas, though entorhinal cortical damage was minimal in this experiment. Furthermore, as indicated earlier, the very extensive dorsal hippocampal lesions, by sectioning the fimbria, completely sever one of the major efferent connections from CA3, whose pyramid cells contribute axons to the fimbria from locations all along the longitudinal axis of Amm o n ' s H o r n 1'14. Thus even when some tissue remained intact near the temporal pole of the hippocampus, it would have been subjected to significant de-afferentation and de-efferentation. Since previous reports have indicated that either 'ventral '26 or 'dorsal '29 hippocampal lesions can abolish blocking, albeit in different species and using very different conditioning paradigms, it is not obvious why extensive lesions including tissue from almost the entire longitudinal axis of the hippocampal formation should have failed to do so. Rickert et al. 26 failed clearly to abolish blocking by making dorsal hippocampal lesions in rats tested under conditions rather similar to ours, while abolishing blocking with ventral hippocampal lesions. One possibility, then, is that our extensive dorsal/adseptal hippocampal damage somehow reversed the consequences which would otherwise follow extensive lesions in the ventral/temporal parts of the structure. As discussed earlier, there is no obvious reason why the hippocampus should be divided into a dorsal and a ventral component, but if extension of our lesions further adseptally abolished the effects of our temporal lesions, this might be a result of effectively severing the hippocampus from the septum. This suggestion would however indicate that combining a fornix lesion with an entorhinal lesion might lead to a reduction in the size of any lesion effects seen. What evidence we have on such combined lesions indicates precisely the reverse of this ~8. Furthermore, since lesions restricted to the 'dorsal' hippocampus have also been reported to abolish blocking, this would lead to the unsatisfactory suggestion that combining two behaviourally equivalent lesions within one structure negates the behavioural consequences that either lesion would have on its own.

50 If the suggestion is therefore discarded, the remaining possibilities are that we produced extra-hippocampal damage different from that produced by other investigators, and accidentally precisely reversed the effects of our hippocampal lesions; or that previous investigators have produced different extra-hippocampal damage from ours, which was actually responsible for the behavioural results they attributed to their hippocampal lesions. At present it is impossible to prefer either of these suggestions to the other. Thus it is not clear what differences in our physiological treatments may have been responsible for the different results we report. It therefore seems more likely that the different results we obtained were a consequence of having used different behavioural parameters. There were, of course, numerous differences in experimental procedure between Rickert's studies and ours; and there are at least some features which might help to account for the difference in outcome. In Rickert et al.'s first study of blocking (ref. 24, Experiment 1), for example, hippocampally lesioned and sham-operated animals were divided into two main groups, one pretrained to CS~ (either a tone or a light), the other given no prior training, before both main groups received a series of CS~ + CS2 trials to the tone-light compound. Although hippocampal rats showed as much suppression to CS2 whether they had been pretrained to CS~ or not, the pre-training had resulted in extremely poor suppression to CS~ in the first place: at the end of Stage 1. hippocampal rats had suppression ratios to CS~ of the order of 0.30, while sham-operated animals had suppression ratios of 0.10. Other things being equal. a failure to condition to CSl can only reduce the extent to which CS~ will block CSe. In Experiment 2 of this paper, an attempt was made to ensure better conditioning to CS~ either by increasing the length of Stage 1 or by increasing the intensity of the shock. Only the first of these procedures was clearly successful, for the stronger shock still left hippocampal animals with a suppression ratio of nearly 0.30 to CS~ at the end of Stage 1 in contrast to a ratio of just over 0.10 in the sham-operated animals. There is a further problem with this second experiment.

however, for it did not include any control groups given no pretraining to CS~. The claim that hippocampal lesions disrupted blocking is based solely on the observation that pretrained hippocampal animals showed more suppression to CS~ on test than did pretrained sham-operated animals. A subsequent paper 2~ relied on a similar comparison between sham-operated and lesioned groups, both given pretraining to CS~ to support the conclusion that ventral hippocampat lesions disrupted blocking. Once again, the lesioned animals showed substantially more suppression to CS~ on test than did the sham-operated animals (0.17 vs 0.46); but they also showed rather more suppression to the C S j - C S , compound at the end of Stage 2 (0.08 vs 0.20} as well as rather tess suppression to CS~ alone at the end of Stage 1 (0.35 vs 0.22). The conclusion that the lesion disrupted blocking would have been very much more secure if control groups given n~ prior training to CS~ had been included and if lesioned and shamoperated animals had shown similar levels of suppression during the course of conditioning. The results from animals given lesions to dorsal hippocampus do little to alleviate one's doubts. Dorsal lesions did not attenuate blocking: in other words, dorsally lesioned rats showed little suppression to CS~ on test. But. unlike animals with ventral lesions, they also showed relativel~ little increase in suppression from the end of Stage 1 to the beginning of Stage 2 on the introduction of CS,. In dorsally lesioned animals the increase in suppression was from 0.35 to 0.29. in animals with ventral lesions the comparable scores were 0.35 and 0.10. It does not seem entirely surprising that the latter group showed more suppression to CS~ tested alone. Indeed. in a preliminary experiment on blocking, with 4 subjects per group and a weaker shock 10.5 mA. 0.5 s), we observed exactly this pattern: hippocampally damaged animals showing less suppress~on at the end of Stage 1 ~mean suppression ratio. 0.19) than cortical or sham controls (0.06 and 0.05. respectively), an increase of suppression at the beginning of Stage 2 (0.19 to 0.06). and more suppression to CS~ when tested alone than cortical or sham controls. There is one further feature of the procedure

51 employed in all 3 of these experiments that differs from that employed in our experiments: animals received no unreinforced pre-exposure to the stimuli used as CSs. This means, in effect, that the first and only occasion on which CS2 was presented on its own was on the critical test trials at the end of Stage 2. In our experiment, as in Kamin's original work on blocking 9, animals received some pre-exposure to CS2 (and to C S 0 before conditioning. Such prior exposure should attenuate any unconditioned suppression produced by a novel CS, and may also reduce generalization from the compound to either CS alone or from one CS to the other. We do not know whether this could have been responsible for the difference in outcome between Rickert's experiments and ours, but it is worth pointing to such an obvious procedural difference. This final point applies equally to Rickert et al.'s experiment on overshadowing 25. Here too, animals received only two test trials to the light at the end of training and here too this was the first occasion that the light had been presented on its own. The other main difference between this experiment and our own Experiment I is that Rickert et al.'s animals appear to have received considerably more training to the TL compounds before being tested. The actual amount of training given is not stated, but it continued either until the animals in the correlated condition reached a criterion of successful discrimination (more than 50°.o suppression to the reinforced compound combined with no suppression to the unreinforced compound) or for 50 sessions (288 trials). It is not stated whether hippocampal and shamoperated animals differed in the amount of training they received, but it does seem likely that there is a very substantial difference between our experiment and theirs in the amount of conditioning received before animals were tested. Whether this is important, we cannot say. Another, more recent experiment on conditioned suppression in rats has also reported that hippocampal lesions attenuate overshadowing 27. In this study, animals with hippocampal, cortical or sham operations were assigned to one of 3 conditions. A control group was exposed to olfactory and visual stimuli in the absence of any shock; a

visual group learned a discrimination between two visual stimuli where neither stimulus was perfectly correlated with the occurrence of shock, although one was a better predictor than the other; an overshadowing group was exposed to exactly the same contingencies between the visual stimuli and shock as the visual group, but in their case the occurrence of shock was perfectly predicted by the odour present on each trial. Only the hippocampal animals showed suppression to the less favourable visual stimulus in this last condition, and the authors interpreted this as evidence of an abolition of overshadowing by hippocampal lesions. There are, however, some problems with this experiment, only some of which are acknowledged by the authors. The hippocampal animals, alone of the 3 groups, appear to have initially preferred the olfactory stimulus correlated with shock in the overshadowing condition. This will presumably have made the olfactory discrimination harder for them than for other animals. There is also evidence, in the visual condition, that the hippocampal animals learned the visual discrimination more rapidly than either of the other groups. Thus the potentially overshadowing discrimination was more difficult, and the potentially overshadowed discrimination less difficult, for hippocampal animals than for sham or cortically lesioned animals. Since there is good evidence that overshadowing increases as the overshadowing cue becomes more salient or the overshadowed cue less salient (e.g. ref. 12), it is not surprising that hippocampal lesions attenuated overshadowing. When considering the conflict between other experimental results and our own, one can argue either that one or other set of results is basically mistaken, or that both should be accepted as valid findings obtained under critically different circumstances. We have suggested, in effect, that Rickert et al.'s experiments on blocking are not wholly convincing: they contain several features that might account for the apparent absence of blocking in animals with hippocampal lesions. The best reason for accepting their results at face value is that an apparently analogous experiment on nictitating membrane conditioning in rabbits has provided clear evidence that lesions to dorsal

52 hippocampus can abolish blocking 29. The experiment seems entirely convincing and we have no wish to dispute the conclusion. But it should not necessarily be generalized to the case of conditioned suppression in rats. If one does wish to make the generalization and, by implication, accept Rickert et al.'s conclusions, this can only be reconciled with the resuits of our experiment by arguing that hippocampal lesions can abolish blocking (perhaps when the effect is small or labile in control animals), but do not necessarily do so. The implication would be that the blocking effect observed in our Experiment II was particularly robust and thus impervious to disruption. This does not seem entirely plausible. Although statistically significant, it was not numerically large (at least by comparison with the effect originally reported by Kamin9); and our control for blocking (prior conditioning to a different auditory CS in Stage 1)was actually a very conservative one which may have reduced the size of the effect. We did, perhaps, ensure a reasonably robust blocking effect by giving 18 trials of conditioning to CS~ in Stage 1 - by comparison with the 12 trials typically provided in Rickert et al.'s experiments. But Rickert et al. (Ref. 24, Experiment II) gave 24trials of conditioning to CS1 and still found no blocking in hippocampally lesioned rats, so this does not seem to be a critical variable. And the main difference between our Experiment I and Rickert et al.'s experiment on overshadowing 25 seems to have been that they provided m o r e discriminative training for their correlated group before testing for control by the common elements. According to most theories (e.g. ref. 23) this should have increased the magnitude of their overshadowing effect and thus made it less sensitive to disruption. Be this as it may, we believe that one conclusion from our experiments is inescapable. Even after large lesions that have destroyed most of the hippocampal formation, rats can still show significant blocking and overshadowing. This conclusion in no way depends on accepting a null result. The stronger conclusion, that hippocampal lesions do not affect blocking or overshadowing, although entirely consistent with our findings,

does require acceptance of the null hypothesis and certainly appears to conflict with other published experiments. We believe, however, that at least in the case of rats and conditioned suppression, it is a possibility which should be taken seriously. ACKNOWLEDGEMENTS

This research was supported by grants from the Science Research Council and the Medical Research Council of Great Britain, awarded to N.J. Mackintosh and J.N.P. Rawlins, respectively

REFERENCES I Andersen. P.. Bland. B.H. and Dudar. J.D.. Orgamzation of the hippocampal output, Exp. Bra#7 Res.. 17 (1973) 152-168. 2 Berger. T.W. and Orr. W.B.. Hippocampectomy selectively disrupts discrimination reversal conditioning of the rabbit nictitating membrane response~ Behav. Brain Res., 8 (1983) 49-68. 3 Blackstad. T.N., Brink. K., Helm. J. and Jeune. B., Distribution ofhippocampal mossy fibres in the rat. An experimental study with silver impregnation methods. J. comp. Neurol.. 138 ( 1970 ) 433-450. a Cotton. M.M.. Goodall, G. and Mackintosh. N.J., Inhibitory conditioning resulting from a reduction in the magnitude of reinforcement, Quart. J. exp. Psychot., 34 (1982) 163-18t. 5 Douglas. R.J.. The hippocampus and behavior. Psvchot. Bull.. 67 (t967) 416-442. 6 Douglas. R.J.. Pavlovian conditioning and the brain. In R.A. Boakes and M.S. Halliday (Eds.), Inhibition and Learning, Academic Press. London. 1972. pp. 529-553. 7 Hjorth-Simonsen. A., Hippocampal efferents to the ipsilateral entorhinal area: an experimental study in the rat. J. comp. Neurol., t42 (197t) 417-437. James. J H . and Wagner. AR.. One-trial overshadowing: evidence of distribution processing, J. exp. Psychol: Anon. Behav. Proc., 6 (1980) 188-205. 9 Kamin. kJ.. "Attention-like' processes m classical conditioning. In M.R. Jones (Ed.), Miami Symposium on the Prediction o f Behavior: A versive Stimulation, University of Miami Press. Miami, 1968, pp. 9-33. l0 Lubow. R.E.. Latent inhibition. P~ychot. Bull., 79 (1973) 398-407. 11 Mackintosh, N.J., A theory of attention: variauon in the associability of stimuli with reinforcement, Psychol. Rev.. 82 (1975) 276-298. 12 Mackintosh. NJ.. Overshadowing and stimulus intensity, AnOn. Learn. Behav., 4 (1976) 186-192.

53 13 Mackintosh, N.J. and Reese, B., One-trial overshadowing, Quart. J. exp. Psychol., 31 (1979) 519-526. 14 Meibach, R.C. and Siegel, A., Efferent connections of the hippocampal formation in the rat, Brain Res., 124 (1977) 197-224. 15 Mitchell, S+J., Rawlins, J.N.P., Steward, O. and Olton, D.S., Medial septal area lesions disrupt theta rhythm and cholinergic staining in medial entorhinal cortex and produce impaired radial arm maze behavior in rats, J. Neurosci., 2 (1982) 292-302. 16 Moore, J.W., Information processing in space-time by the hippocampus, Physiol. Psychol., 7 (1979) 224-232. 17 Moore, J+W. and Stickney, K.J., Formation of attentiohal-associative networks in real time: role of the hippocampus and implications for conditioning, Physiol. Psychol., 8 (1980) 207-217. 18 Olton, D.S., Walker, J.A. and Gage, F.H., Hippocampal connections and spatial discrimination, Brain Res., 139 (1978) 295-308. 19 Pavlov, I.P., Conditioned Reflexes (trans. G.V. Anrep), Oxford University Press, London, 1927. 20 Pearce, J.M. and Hall, G., A model for Pavlovian learning: variations in the effectiveness of conditioned but not of unconditioned stimuli, Psychol. Rev., 87 (1980) 532-552. 21 Rawlins, J.N.P., Feldon, J. and Gray, J.A., Septo-hippocampal connections and the hippocampal theta rhythm, Exp. Brain Res., 36 (1979) 49-63. 22 Rawlins, J.N.P. and Bennett, R.C., A headholder for visually guided surgery in rats, Physiol. Behav., 24 (1980) 415-416. 23 Rescorla, R.A. and Wagner, A.R., A theory of Pavlovian conditioning: variations in the effectiveness of reinforcement and nonreinforcement. In A.H. Black and W.F. Prokasy (Eds.), Classical Conditioning H: Current Research and Theory, Appleton Century Crofts, New York, 1972, pp. 64-99.

24 Rickert, E.J., Bennett, T.L., Lane, P.L. and French, J., Hippocampectomy and the attenuation of blocking, Behav. Biol., 22 (1978) 147-160. 25 Rickert, E.J., Lorden, J.F., Dawson, R., Smyly, E. and Callahan, M.F+, Stimulus processing and stimulus selection in rats with hippocampal lesions, Behav. neural. Biol., 27 (1979) 454-465. 26 Rickert, E.J., Lorden, J.F., Dawson, R. and Smyly, E., Limbic lesions and the blocking effect, Physiol. Behav., 26 (1981) 601-606. 27 Schmajuk, N.A., Spear, N.E. and Isaacson, R.L., Absence of overshadowing in rats with hippocampal lesions, Physiol. Psychol., 11 (1983) 59-62. 28 Schmaltz, L.W. and Theios, J., Acquisition and extinction of a classicallyconditioned response in hippocampectomized rabbits ( Orytolagus cuniculus ). J. comp. physiol. Psychol., 79 (1972) 328-333. 29 Solomon, P.R., Role of the hippocampus in blocking and conditioned inhibition of the rabbit's nictitating membrane response, J. comp. physiol. Psychol., 91 (1977) 407-417. 30 Solomon, P.R. and Moore, J.W., Latent inhibition and stimulus generalisation of the classically conditioned nictitating membrane response in rabbits following dorsal hippocampal ablation, J. comp. physiol. Psychol., 89 (1975) 1192-1203. 31 Wagner, A.R., SOP: a model of automatic memory processing in animal behavior. In N.E. Spear and R.R. Miller (Eds.), Information processing in animals. memory mechanisms, Erlbaum, Hillsdale, NJ, 1981, pp. 5-47. 32 Wagner, A.R., Logan, F.A., Haberlandt, K. and Price, T., Stimulus selection in animal discrimination learning, J. exp. Psychol., 76 (1968) 171-180. 33 Weisz, D.J., Solomon, P.R. and Thompson, R.F., The hippocampus appears necessary for trace conditioning, Bull. Psychol. Soc., 16 (1980) 164 (Abstract 194).