Neurotoxic lesions of the perirhinal cortex do not mimic the behavioural effects of fornix...

E L S E V I E R Behavioural Brain Research 80 (1996) 9-25

BEHAVIOURAL BRAIN

RESEARCH

R e s e a r c h r e p o r t

Neurotoxic lesions of the perirhinal cortex do not mimic the behavioural effects of fornix transection in the rat

A b d e l k a d e r E n n a c e u r a , , , N i c k N e a v e u, J o h n P. A g g l e t o n c

a university of Durham, Department of Psychology, Science laboratories, South Road, Durham, DHI 3LE, UK b University of Northumbria, Division of Psychology, Ellison Place, Newcastle upon Tyne, NE1 8ST, UK

c School of Psychology, University of Wales College of Cardiff, PO Box 901, Cardiff, CF1 3 YG. UK

Received 29 May 1995; revised 8 December 1995; accepted 13 December 1995

Abstract

The effects of perirhinal (Prh) and fornix (Fx) lesions were compared on a series of spatial and nonspatial memory tests. These tests included delayed nonmatching-to-position in an operant chamber, a spatial (lever) discrimination and its subsequent reversals, delayed spatial alternation in a T-maze, and an object recognition memory test using both normal objects and 're configured' objects. As expected, the rats with fornix lesions were impaired on all of the spatial tests. Their performance on the recognition test was, however, left intact. The perirhinal lesions produced a quite different pattern of results. Animals with these lesions were unimpaired on all three spatial tasks, but displayed evidence of an impairment on the object recognition test. This impairment was restricted to the longer delay (15 rain) and was only found with the normal objects. These findings suggest that the actions of the perirhinal cortex and the hippocampus can be dissociated from one another.

Keywords: Entorhinal cortex; Hippocampus; Spatial memory; Object recognition; Configural association; Learning; Memory; Rat

1. Introduction

Research over the last few years has led to an important reappraisal of the contributions of the hippocampus and adjacent cortical regions to memory. Much of the evidence for this reappraisal has come from selective lesion studies in monkeys [25,39,45,64,72,73] and from recordings of single-unit activity in the primate temporal lobe [7] . Further evidence has come more recently from behavioural and electrophysiological studies of rats [27,31,41-43,38,48,50,71]. The vital step that triggered this advance was to distinguish the contributions of the hippocampus and subiculum from those of the entorhinal, perirhinal, and parahippocampal cortices. From such studies it now appears that the rhinal and parahippocampal cortices are of enormous importance for object recognition while the contribution of the hippocampus may be relatively minor [25,34,39,45,60]. It therefore seems that the hippocampus is specialised for only certain classes of information (e.g., spatial memory).

* Corresponding author. Fax: (191) 374 74 74; E-mail: [email protected]

0166-4328/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0166-4328 (96)00006-X

These findings have led to a number of proposals concerning the functional relationship between the hippocampus and the adjacent rhinal regions. One prevalent view is that these two regions form a unitary memory system, in which the rhinal cortical regions form not only a vital pathway for information reaching the hippocampus but also subserve other memory functions [13,60]. As a consequence, the rhinal region is thought to be important for a broad array of memory tasks, including object recognition, while only a subset of these tasks depend on hippocampal activity [13,60]. This view accords with the pattern of anatomical interconnec- tions between these two regions [9,54,65,66,69]. One testable prediction is that the deficits associated with hippocampal and rhinal lesions can be dissociated from one another, but that any dissociation will only occur in one direction (rhinal lesions will produce more severe impairments than hippocampal lesions).

The present study sought to test this prediction in rats by comparing the behavioural effects of lesions in the perirhinal cortex with those of fornix lesions, The perirhinal region was selected as a recent study of

10 Abdelkader Ennaceur et al./Behavioural Brain Research 80 (1996) 9-25

monkeys has show that, within the rhinal region, damage to the perirhinal cortex is sufficient to produce a severe recognition memory deficit on the delayed nonmatching- to-sample task [34]. There is similar evidence suggesting that perirhinal lesions in rats can also impair recognition memory [38,48]. Comparisons were made with lesions of the fornix, rather than the hippocampus. This target was selected as it is well established that cutting this tract produces a very similar array of deficits to those produced by hippocampectomy [3,24,26]. A particular advantage in targeting the fornix is that this region can be lesioned with no risk of direct damage to the rhinal cortex. Finally, it should be noted that the perirhinal lesions were made by injecting the neurotoxin N-methyl- D-aspartic acid (NMDA). This method differs from the majority of previous studies which have used aspiration. The rationale for this approach stemmed from the desire to minimise damage to underlying white matter that might contribute to any resultant lesion effect [41].

The first behavioural test was an automated delayed nonmatching-to-position (DNMP) task. This task was selected because it is sensitive to hippocampal damage and to damage in certain brain regions closely related to the hippocampus [24,10,11 ]. The effects of perirhinal damage upon this task have not, however, been described. The DNMP task was followed by a lever discrimination with subsequent reversals, and a rewarded spatial alternation task in a T-maze. The performance of these latter two tasks is also known to be disrupted by hippocampal or fornix damage [ 1,6,26]. In addition, the animals were assessed on a test of object recognition that uses differential levels of exploration between familiar and unfamiliar objects as a behavioural measure for recognition [15,20]. This task is based on rat's spontaneous preference for novelty and has demon- strated sensitivity to various drugs and lesions treatment [8,14,16,18,19,28,35,58,70]. This task was selected in response to the severe delayed nonmatching-to-sample deficits associated with rhinal lesions in monkeys. The rats were also tested on their ability to distinguish between two stimuli that were composed of the same elements, but in one case the elements were spatially rearranged to form a novel configuration. This followed from the proposal that the hippocampus is required for the creation of configural associations [63].

2. Materials and methods

All rats were trained prior to surgery on the DNMP task to a standard criterion level. Following surgery and recovery they were tested in the following order: (1) retest of the DNMP task; (2) reversals of a lever (spatial) discrimination task in an operant chamber; (3) spontaneous object recognition test in an open box; and (4) spatial forced-alternation in a T-maze.

Subjects. The study involved 31 naive, male rats of the pigmented DA strain (Bantin and Kingman, Hull). Throughout the period of the experiment the animals were housed individually under diurnal conditions (14-h light/10-h dark), all testing occurring at a regular time during the light period. The animals were tested for 5 days a week, and prior to test days fed approx. 15 g of RM1 laboratory diet (Special Diet Services, Witham, Essex) daily so that they did not drop below 85% of normal body weight. At the start of testing the animals were aged 4 months, and they weighed between 250 and 270 g at the time of surgery. All animals had free access to water.

Surgical and histological procedures. A total of 31 rats received surgeries. These were divided into three groups; perirhinal cortical lesions (Prh, n = 9), fornix lesions (Fx, n= 8) and surgical controls (Co, n = 14). Prior to surgery all animals were deeply anaesthetised by intraperitoneal injection (60 mg/kg) of pentobarbitone sodium and then placed in a stereotaxic headholder (David Kopf Instruments, Tujunga). The scalp was then retracted to expose the skull. Craniotomies were then made directly above the target regions, and the dura cut to expose the cortex.

For the perirhinal cortical lesions (Prh), injections of 0.28 gl of 0.09 M N-methyl-D-aspartic acid (NMDA) (Sigma Chemical Co., Poole, UK) dissolved in phosphate buffer (pH 7.2) were made through a 1 gl Hamilton syringe into three sites in each hemisphere. Each injection was made gradually over a 3-min period and the needle was left in situ for a further 3 min before being withdrawn. The stereotaxic co-ordinates relative to ear- bar zero, with the incisor-bar set at + 5.0 relative to the horizontal plane were: AP + 3.9, LAT +_ 5.8, HT + 3.2; AP +1.9, LAT +6.0, HT +1.6; and AP +0.2, LAT ± 6.3, HT + 2.4.

The initial stages of the fornix lesions (Fx) were the same as those described above for the Prh lesions, but the actual lesion was made by radiofrequency. A Radionics TCZ (Radionics Inc., Burlington) electrode (0.3 mm tip length and 0.25 mm diameter) was lowered vertically into the fornix and the tip temperature raised to 75°C for 60 s using an RFG4-A Lesion Maker (Radionics Inc., Burlington). Two lesions were made in each hemisphere. The stereotaxic co-ordinates of the lesion relative to ear-bar zero, with the incisor bar set at +5.0, were: AP +5.3, HT +7.1 LAT ±0.7 and AP +5.3, HT +7.1 LAT +1.6.

For the surgical control animals (Co) the procedure was essentially the same as that for the above surgeries except that a single craniotomy was made over the midline and the dura cut in both hemispheres as for a Fx lesion. At the completion of all surgeries sulphanila- mide powder was applied and the skin sutured.

On completion of the experiment the animals were killed with an overdose of Nembutal and perfused

Abdelkader Ennaceur et al./Behavioural Brain Research 80 (1996) 9-25 l 1

intracardially with 5% formol-saline. The brains were then rapidly removed and placed in 5% formol-saline. Subsequently, the brains were blocked, embedded in wax (Paraplast), and cut into 10 pm coronal sections. Every tenth section was mounted and stained with cresyl violet, a Nissl stain.

2.1. Experiment 1 and2: Performance of rats in DNMP and Spatial Discrimination tests

Apparatus. The delayed nonmatching-to-position task (Experiment 1), and the discrimination and reversal task (Experiment 2) were both carried out using 7 operant chambers (Campden Instruments Ltd., Loughborough) under the control of 2 Spider microprocessors (Paul Fray Ltd., Cambrifge). Each chamber was fitted with two retractable levers situated 7.5 cm either side of a central food tray. The magazine tray assembly, which delivered 45 mg pellets (Campden Instruments, Loughborough), had a hinged perspex lid at which nose pokes could be recorded. A light was located inside the food tray and a house light was located in the centre of the roof. A further light was positioned above each lever.

Procedure in the delayed nonmatching-to-position (DNMP) test. All animals received magazine training followed by an autoshaping procedure that trained the rats to respond on both levers [56]. Following this, D N M P training began. By the end of training all rats would respond to the following sequence of events. After a 10-s inter-trial interval, either the left or the right hand lever emerged, and the stimulus light above it was simultaneously illuminated to provide an extra cue. The rat had to respond to this sample lever (within 10 s), upon which the lever was retracted and the magazine tray illuminated. The subject now had to approach the tray and repeatedly operate the magazine flap (nose- poke). The first nose-poke after a predetermined 'retention' delay caused both levers to emerge and the stimulus lights above them to be illuminated. If the rat responded on the lever that had not been the sample (nonmatch) it was rewarded with a 45 mg food pellet. Incorrect responses, or a failure to respond, resulted in a 'time- out' of 10 s, during which all of the lights in the chamber were switched off and both levers retracted; these were the only occasions when the house light was unlit.

During the initial training procedure there was no retention delay following a response to the sample lever, but following a pretraining period, a variable delay was placed between the sample and test phases. By requiring the rat to respond on a central panel during this retention delay it was intended to restrict the use of mediating strategies. The training procedure prior to surgery consisted of a number of stages of increasing difficulty, each of which had their own criterion [44]. These culminated in the use of retention delays of up to 32 s.

Each session consisted of 96 trials, and for the final

six sessions before surgery there were an equal number of retention delays of 0, 2, 4, 8, 16 and 32 s. These delays were presented in a balanced order. Following this all rats received either fornix, rhinal or control (sham) surgeries. Approx. 6 weeks after surgery, all animals were retested on the D N M P task. The first seven sessions used only 'O's delays (i.e., until the first nose-poke), while the next two sessions used delays of 0, 2 and 4 s. These were followed by 15 sessions which contained a balanced mixture of 0, 2, 4, 8, 16 and 32 s delays over 96 trials. Data analyses were carried out on this final series of 15 sessions.

Performance measures and analyses. The data analyses provided three indices of accuracy. These consisted of percent correct, and two measures of sensitivity (A' and SI) derived from nonparametric signal detection (TSD) models [22]. Three measures of bias (B', RI and Iy) were also calculated, the first (B') is thought to reflect perceptual bias, while the second (RI) is a measure of response bias. A third measure (Iy) contrasts accuracy between the two levers [22,57]. Finally, estimates of general responsivity were also recorded. These included (a) latency to respond to the sample lever, (b) latency to make the first magazine response ('nose-poke'); (c) rate of responding to the magazine flap, or nose-pokes/s, excluding the 0 s delay condition; (d) choice (and average choice) latencies; and (e) the number of missed trials. The justification and calculation of these measures and indices has been described elsewhere [2,57].

Prior to analysis all data were transformed as appro- priate (arcsin: all accuracy and bias indices; logarithmic: all latencies; square-root: misses), and analysed by para- metric analysis of variance (ANOVA) including one- factor (lesion group) independent measure and two- factor (lesion group, delay) mixed measures analysis. When the F-ratios were significant (P <0.05), the means were compared using the Newman-Keuls procedure [68].

Procedure in the spatial discrimination and reversal tests. One week after completing the DNMP task, all animals were trained on a spatial discrimination task in the same operant chambers 1-44]. For each session the animal was presented with both levers, one of which had been arbitrarily designated as S + and the other as S - . The levers remained extended until one was pressed (there were no 'miss' trials) at which time both levers were retracted. Responding on the correct (S+) lever was followed by the delivery of one reward pellet. Responding on the incorrect lever ( S - ) was recorded as an error and no reward was given. Following an intertrial interval of 7 s the sequence was repeated. Each session consisted of 40 trials, the correct lever remaining on the same side during a given session. All animals received 20 sessions, the position of the correct lever following a balanced, pseudorandom sequence. This meant that a session could either be 'consistent' (i.e., the S+ lever


was on the same side as on the previous session) or it could a 'reversal' (i.e., the S + lever was on the opposite side).

Performance measures and analyses. Initial statistical analysis were performed on the mean percent correct responses over the first two sessions, in which the S + was consistent, i.e., the initial lever discrimination. The scores over the remaining 18 sessions (combined reversal and consistent) were then compared. By excluding the first session, the scores could also be divided between those nine sessions where the correct lever was in the same position as in the previous session (consistent), and those 10 sessions where the correct lever reversed sides between sessions (reversal). These were then compared. A final set of analyses considered only the first 10 trials of each session. This was done in order to focus on those trials where the effects of the previous session might be expected to be most apparent, and to avoid possible problems from ceiling effects.

a) Object recognition test: standard condition

4, m

¢ b) Object recognition test: eonfigural condition I

2.2. Experiment 3." Performance of rats & a spontaneous object recognition test

Apparatus. The apparatus consisted of an open box (100 x 100 × 50 cm) made of aluminium, the inside of which was painted grey. The floor was covered with sawdust. Triplicate copies were made of the objects to be discriminated, which were made of glass, plastic or metal. The weight of the objects ensured that they could not be displaced by the rats. The objects used in one session are never reused in another session.

Behavioural testing. All rats were given five habituation sessions where they were allowed 3 rain to explore the apparatus (with no objects present). Forty-eight hours later, testing began. In none of these tests did the experimenter know the identity of the animals. Testing began approx. 16 weeks after surgery.

Rats were first tested on the standard condition (Experiment 3a; Fig. la) with retention delays of 1 min and 15 min. After that they were tested on the 'configural conditions' (Experiment 3b; Fig. lb,c) using retention delays of either 1 or 15 min. Each rat received one session on each condition, and the interval between sessions was 48 h. A session consisted of a sample phase and a choice phase, with an intervening delay period. The duration of each phase was 3 min.

At the start of each sample phase two identical objects (A1 and A2) were placed in the back corner of the box, I0 cm from the side wall. A rat was then placed in the middle of the box and the total time spent exploring the two objects was determined from video taped recordings of each session. Exploration of an object was defined as directing the nose to the object at a distance of less than 2 cm and/or touching it with the nose. Turning around or sitting on the object was not considered as exploratory behaviour. After a delay the rat was reintroduced to the

N /

c) Object recognition test: conflgural condition 2

Fig. 1. Standard and configural object recognition testing conditions. Left (sample phase), rats are exposed to two identical objects. Right (choice phase): (a) in the standard condition (A vs. B), rats are exposed to two different objects, the sample previously explored in the sample phase which is now familiar (A) and a new object (B), never seen before; (b) in the first configural condition (A vs. A*), rats are exposed to two different objects, the sample previously explored in the choice phase which is now familiar (A) and a re-confguration of the sample object (A*); (c) in the second configural condition (A* vs. B), rats are exposed to two different objects, the re-configuration of the sample object (A*) and a new object (B), never seen before.

box. In the standard condition the box now contained a third identical copy of the familiar object (A3) and a new object (B). These were placed in the same locations as the sample stimuli. The role (sample, new, or re-configured), as well as the location of the two choice objects, was counterbalanced between rats. As far as could be ascertained the objects had no natural significance for the rats and they had never been associated with a reinforcer.

The configural conditions were the same as those used in the standard condition, except that now a third identical copy of the sample stimulus (A) was re-configured for the choice phase. A re-configured stimulus (A*) consisted of a different spatial arrangement of the elements of the original sample (A). This meant that

Abdelkader Ennaceur et al./Behavioural Brain Research 80 (1996) 9-25 13

although each constituent part of the sample was familiar, the overall appearance was novel. Care was taken not to introduce any hidden aspect of the sample object (e.g., the base) when it was rearranged as a re-configured object.

Two different 'configural' tests were employed. In the first condition (Fig. lb), the original sample (A) was presented in the choice phase with its own re-configured version (A*). In the second condition (Fig. lc), the rat was presented in the choice phase with the re-configured version of the sample (A*) and a new object (B). New sample stimuli were used for every session.

Performance measures and analyses. The basic measure was the time spent by the rats in exploring objects during the sample phase and during the choice phase. Exploration of an object was defined as follows: directing the nose to the object at a distance <2 cm and/or touching it with the nose; turning around or sitting on the object was not considered as exploratory behaviour.

Comparisons focused on the total time spent exploring objects during the sample and choice phases. Analyses of variance were performed on the following measures (Table 1): ( 1 ) el, which is the total time spent in exploring the two identical objects in the sample phase; (2) e2, which is the total time spent exploring the two objects in the choice phase; (3) dl , the discrimination index, which is the difference in time spent exploring the two objects in the choice phase (e.g., B - A in the standard condition, or A* - A and B - A * in the configural conditions); and (4) d2, the discrimination ratio, which is the difference in exploration time (d l) divided by the total time spent exploring the two objects in the choice phase, e.g., B - A / A + B in the standard condition, and A * - A / A * + A or B - A * / B + A* in the configural conditions. This ratio makes it possible to adjust for any differences in the total amount of exploration time.

To confirm whether an individual group had discriminated between two objects, paired Student t-tests (one- tailed) were used to compare the time spent exploring each of the objects in the choice phase of the various

Table 1 Index of the different measures involved in the object recognition test: el is the measure of the total time spent exploring the two objects A1 and A2 in the sample phase, whereas e2 is the measure of the total time spent exploring both objects B and A3, A3 and A*, or A* and B in the choice phase. A3 is the copy of the sample which is now familiar, A* is the configuration of the sample, and B is the new object, d l is the discrimination index (difference in time spent exploring the two objects in the choice phase) and d2 is the discrimination ratio (difference of exploration time divided by the total time spent exploring the two objects in the choice phase)

Variables el e2 dl d2

Condition 1 A1 + A2 A3 + B B - A 3 B - A 3 / B + A3 Condition 2 AI + A2 A3 + A* A * - A 3 A * - A 3 / A * + A3 Condition 3 A1 + A2 A* + B B - A * B - A * / B + A*

conditions. The threshold level for significance was P ~ 0.05.

2.3. Experiment 4: Spatial forced-alternation

Apparatus. For the forced-alternation task the animals were tested in a T-maze. The floors of the T-maze were 10 cm wide and made of aluminium. The stem was 80 cm long with a guillotine door located 33 cm from the beginning. The cross piece was 136 cm long and at each end there was a food well 4 cm in diameter and 0.75 cm deep. The walls of the maze were 17 cm high and made of clear Perspex. The maze was supported on two stands 93 cm high. Lighting was provided by fluorescent lights suspended 92 cm above the apparatus, the luminance light levels at the choice point and food wells being 32 lux and 28 lux, respectively.

Procedure in the spatial forced-alternation test. Testing began 8 weeks after completion of Experiment 2. Each animal was given 1 or 2 days of pretraining in order to run reliably down the stem of the maze to find food pellets in the food wells in both arms. Following this the experiment began. At the start of each trial, which consisted of two stages, three food pellets (45 rag; Campden Instruments, Loughborough), were placed in each food well and a wooden block was placed at the neck of the T-maze so closing off one arm. As a consequence, the animal was forced to enter a pre selected arm on each 'sample run' and then allowed to eat the food there. The animal was then picked up and confined in the start box for a delay of 15s, during which the wooden block was removed. The door to the start box was then opened and the animal allowed a free choice between the two arms of the T-maze. On this 'choice run' the criteria for arm selection was placing a back foot in one of the arms. No retracing was permitted. If the rat had alternated, i.e., had entered the arm not previously visited on the 'sample run', it was allowed to eat the food reward before being returned to its cage. If the other arm was chosen, i.e., the same arm as visited on the 'sample run', the rat was confined to that arm for approx. 10 s, and then returned to its cage.

The rats were tested in groups of three or four with each rat having one trial in turn, so that the intertrial interval was about 4 min, The animals received six trials a day, for a total of six sessions. This was immediately followed by a further 10 sessions in which the six trials were divided equally between those with retention delays (i.e., the time spent in the start box prior to the 'choice run') of either 10, 30 or 60 s, making a total of 20 trials at each delay.

Performance measures and analyses. ANOVA and subsequent Newman-Keuls tests were performed on the mean percent correct scores in the initial six acquisition sessions with a retention delay of 15 s, and on the mean percent correct scores for the 20 trials at each of three


delays (10, 30 and 60 s). Finally, a comparison using the total scores taken over both the acquisition and delay conditions was performed in order to utilise all alternation data.

3. Results

Histological analysis. Following histological analysis, one animal in the Co group was excluded due to vascular damage in the cortex. The fornix lesions produced very extensive damage to the fibre tract itself and in most cases also extended into the most rostral head of the hippocampus. In six of the eight animals the tract was completely severed bilaterally, while in the remainder only the most lateral tips of the fimbria were spared (Fig. 2). In nearly all cases the lesion, in addition to damaging a small part of the corpus callosum, involved the very most dorsal margin of the anterior thalamic nuclei. The cingulum bundle was always spared and there was no direct damage to the cingulate cortex.

The Prh lesions were consistently centred in the depths of the rhinal sulcus, extending rostro-caudally from the anterior third of the amygdala to the caudal extent of the hippocampus (Fig. 3a,b). The lesions were therefore centred in the perirhinal cortex, destroying a large proportion of this region. Within the region of the lesion there was little or no evidence of neuronal sparing. In most cases the lesion extended ventrally to include variable amounts of the piriform cortex (rostrally) and the lateral entorhinal cortex (caudally). In those cases with the largest lesions this meant that almost the full extent of the piriform or the lateral entorhinal cortex was involved. The lesions stopped close to the caudal limit of the perirhinal cortex, but as a consequence they consistently spared the most caudal portion of the entorhinal cortex (see Plate 1, photomicrographs). In all cases damage was found in that part of area TE3 adjacent to the perirhinal cortex. A very restricted patch of cell loss was found in all animals in that part of the CA1 field adjacent to the depths of the perirhinal cortex. In six of the nine cases this CA1 damage was only unilateral.

t

Fig. 2. Coronal section illustrating the extent of the largest (horizontal + vertical lines) and smallest (vertical lines) fornix lesions. Abbreviations: D3V, dorsal portion of third ventricle; LV, lateral ventricle.

3.1. Exper imen t 1: De layed nonmatching-to-posi t ion

Following histological analysis, the groups comprised 13 Co, 9 Prh and 8 Fx animals. Comparisons of the percent correct scores on the first seven sessions (0 s delay only) and the next two sessions (0 s and 4 s delay) failed to show a group difference (P>0.10). This showed that all three groups could perform the basic task elements.

D N M P - Accuracy measures. An analysis of variance using the percent correct scores (Fig. 4 and 5) overall subsequent post-operative sessions (delays 0-32 s) revealed a significant group effect (F(2,27)=10.22, P < 0.01). There was also an effect of session (F(14,378) = 3.49, P<0.01), delay (F(5,135)=433.03, P<0.01), and a session x delay interaction (F(70,1890)= 1.39, P < 0.05). The session effects reflected the overall improvement in scores with practice, while the group effects reflected the poor performance of the Fx group. A subsequent Newman-Keuls analysis revealed that neither the Co nor the Prh groups differed from each other, but that both groups differed from the Fx group (P < 0.05).

The same pattern of results was found with the two TSD measures of sensitivity, A' and SI (Fig. 5). Both of these measures not only revealed highly significant effects of session and delay (P <0.01), they also both indicated a significant group effect (A': F(2,27)=8.59, P<0.025; SI: F(2,27)=10.72, P<0.01). As before, a Newman- Keuls analysis revealed that the group effects reflected an impairment in the Fx group, and that this group differed from both the Co and Prh groups (P<0.05). The Co and Prh groups did not differ from each other.

D N M P - Bias indices. As expected from previous studies, all three bias indices (Iy, B" and RI) demon- strated both session and delay effects (P<0.01 in all cases; Fig. 5). These effects reflected a decrease in bias over sessions, but an increase with longer delays (Fig. 5). In addition, each of the bias measurements revealed a significant group effect (Iy: F(2,27)=6.10, P<0.01; B": F(2,27)=5.95, P<0.01; RI: F(2,27)=10.57, P<0.01), and for two of the indices there was also a significant group x delay interaction (B": F(10,135) = 2.77, P < 0.01; RI: F(10,135)=2.07, P<0.05). A subsequent Newman- Keuls analysis showed that for all three measures of bias the significant group effects could be attributed to the Fx group which consistently showed higher levels of bias than both the Co and Prh animals (P<0.05). Similarly, the group x delay interactions reflected the unusually rapid increase in bias scores shown by the Fx animals with increasing delay.

D N M P - General responsivity. There were no group effects for a number of general measures of responsivity. These were mean latency to sample (F(2,27)=1.09, P>0.10), total number of misses ( F < I ) , magazine response (nose-poke) rate (F(2,27)=2.41, P>0.10), and mean latency to first nose-poke (F(2,27)= 2.04, P > 0.10).

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Fig. 3. (a) lateral surface view depicting the extent of common bilateral damage in the two hemispheres of the largest (vertical lines) and smallest (horizontal lines) Prh lesions. The border of the piriform (Pir), perirhinal (Prh) and entorhinal (Ent) cortices are taken from Paxinos and Watson [491; (b) coronal sections illustrating the extent of the largest (vertical lines) and smallest (horizontal lines) cortical damage. The numbers correspond to the approximate position from bregma according to the atlas of Paxinos and Watson [49].

16 Abdelkader Ennaceur et al./Behavioural Brain Research 80 ( l 996) 9-25

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Abdelkader Ennaceur et al./Behavioural Brain Research 80 (1996) 9 25 17

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SESSIONS

Fig. 4. Mean percent correct scores on the DNMP task over each of the 15 postoperative sessions using the results from all six delays. Delay data have been pooled.

Group differences were found, however, for the mean choice latency (F(2,27)=4.82, P<0.05) , which a subsequent Newman-Keuls analysis revealed to be due to the Fx group who responded with a significantly shorter latency than the Co or Prh groups.

did not differ from the Co animals. There were no group differences on the consistent sessions.

A final set of analyses considered only the first 10 trials of each session, when the effects of the previous session might be expected to be most apparent. Figure 7 shows the mean percent correct scores for the first 10 trials on the 'consistent' and the "reversal' sessions. As expected, there was a highly significant effect of type of session (F(1,27)=281.3, P<0.0001), showing how all three groups performed better when the correct lever was in the same position as in the previous session. There was also a significant group effect (F(2,27)= 3.38, P<0.05) , which a subsequent Newman-Keuls test revealed to be the result of the poor performance of the Fx group when compared with the Controls (P<0.05). This lower level of performance was, however, only observed on the 'reversal' sessions. Consistent with this was the highly significant group × type of session interaction (F(1,27)= 17.82, P<0.001).

3.3. Experiment 3." Spontaneous object recognition test

3.2. Experiment 2." Spatial discrim&ations and reversals

The second experiment consisted of a spatial (lever) discrimination task (Sessions 1 and 2) followed by a series of reversals between specified sessions. The mean percent correct responses for the four groups over the 20 sessions were: Co=77.7, Prh=73.7 and Fx=74.3; (Fig. 6). An ANOVA carried out on the initial two discrimination trials revealed no significant differences between the groups (F(2,27)=2.95, P>0.05). Further analysis of the remaining 18 sessions did, however, reveal a significant group difference (F(2,27)=3.39, P<0.05) . A Newman-Keuls test carried out on this result showed that the group difference was due to the poorer performance of the Fx group which differed from the Co group (P<0.05), but did not differ from the Prh group. The Co and Prh groups did not differ from each other.

By excluding the first session, the scores could be divided between those nine sessions where the correct lever was in the same position as in the previous session (consistent), and those 10 sessions where the correct lever reversed sides between sessions (Fig. 7). As expected the scores for the consistent sessions were considerably higher than those for reversal sessions, and this was reflected in a large type of session effect (F(1,27)=288.2, P<0.001). While the mean scores on the 'consistent' sessions were all very similar, a clear difference appeared for the 'reversal' sessions (Fig. 7). An ANOVA revealed that while there was no overall group difference (F(2,27)=2.27), there was a significant interaction between type of session (consistent or reversal) and lesion (F(2,27)= 7.65, P < 0.01). This interaction reflected the particularly poor performance of the Fx group on the reversal sessions, while the Prh animals

Experiment 3a ('Standard version'). It was found that both the Fx and Co groups spent significantly more time exploring the new object than the familiar object, as measured by the difference in time spent exploring the objects in the choice phase (dl). This was found both for the delays of 1 min and 15 rain (all P<0.01). While the Prh group also spent significantly more time exploring the unfamiliar object after a delay of 1 min (ts =5.2, P<0.001), the same animals did not appear to distinguish between the two objects in the choice phase after a delay of 15 min (t8 =0.48, P>0.01).

A further analysis of the exploration behaviour of the three groups used the discrimination ratio (d2) which takes into account individual differences in overall levels of exploration (Fig. 8). It should be added that there were no group differences in the total amounts of exploration time in either the sample phase or the choice phase (both, F < 1). An analysis of variance using d2 for both 1 and 15 rain confirmed that there was a group effect (F(2,27)=3.90, P=0.03), but no delay effect (F(1,27)=2.53, NS) and no group×delay interaction (F(2,27) = 1.66). The group effect primarily reflected the lower levels of discrimination shown by the Prh group for the 15 min delay condition (Fig. 8). As a consequence the Prh group differed from both the Co and Fx groups (P<0.05). Although there was no group ×delay interaction, the discrimination ratio of the Prh group was significantly lower than that of the Co and Fx groups after the retention delay of 15 min (F(1,27)=3.59, P = 0.04), but not after just 1 rain (F(1,27)=0.35).

Experiment 3b ('Configural conditions'). In the first configural condition the animals were offered a choice between the now familiar sample (A) and its rearranged version (A*). All three groups of rats showed that they

18 Abdelkader Ennaceur et aL /Behavioural Brain Research 80 (1996) 9-25

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Fig. 5. DNMP: Accuracy (left column) and bias (right column) indices as a function of lesion. See text for explanation of indices. All scores have been converted to absolute values and read from 0 to 100. In the case of the bias scores, 100 represents a complete bias and 0 neutral, or no bias. (Note different scales on the vertical axis.)

Abdelkader Ennaceur et al. 'Behavioural Brain Research 80 (1996) 9-25 19

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could discriminate the configured stimulus (A*) after a 1 min delay as they spent more time in exploring A* than A (dl, maximum P=0.03). None of the groups, however, showed evidence of discrimination after the 15 min delay. Consistent with this, an analysis of variance using the discrimination ratio (d2) showed a significant delay effect (F(1,27)=9.37, P=0.004), reflecting the

difference between 1 min and 15 min (Fig. 9), but no group effect and no interaction (both F < 1). It should be added that although the overall levels of exploration in the sample phase (el) were comparable between the three groups (F < 1), there was a significant group x delay interaction effect for the total amount of exploration during the choice phase (e2: F(2,27)=3.54, P=0.04).


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This reflected lower levels of exploration by the Prh group during the 15 min choice session. As the overall pattern of results for dl and d2 were the same, and d2 takes into account variations in exploration levels, this minor difference does not appear to have influenced the result.

In the second configural condition the animals were offered a choice between the re configured sample (A*) and a novel object (B). Unlike the other two groups, the Prh animals spent significantly more time exploring the re configured sample than the new object after a delay of 1 min (dl: ts=2.47, P=0.02) . After a delay of 15 min both the Prh and Co groups now spent more time exploring the new object than the re-configured sample (dl, Prh: t 8 =4.30, P=0.001; Co: t12=3.32, P = 0.003). Although the Fx groups also spent more time exploring the novel object the difference was not significant. An analysis of variance using the discrimination ratio (d2, Fig. 10) showed only a delay effect (F(1,27)= 9.65, P -0 .003) . This reflected the change in choice from the re configured object to the novel object. There was no evidence of a significant group effect (F(1,27)=0.52, P>0 .10) or of a g roup×de lay interaction (F(1,27)= 074, P>0.10) . Finally, there was no evidence that any of the three groups differed in the various measures of total exploration time.

3.4. Experiment 4." Spatial forced-alternation

During the initial six acquisition sessions each animal performed a total of 36 trials with a retention delay of

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15 s (Fig. 11). Scores were analysed using an ANOVA which revealed a highly significant group effect (F(2,27) = 59.9, P < 0.001). Subsequent Newman-Keuls tests showed that this reflected the poor performance of the Fx group, which was significantly worse than both


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of the other groups (P<0.001). Neither the Co nor the Prh groups differed from each other.

The mean percent correct scores for the three delays (10, 30 and 60 s) are shown in Fig. 11. There was a highly significant group effect (F(2,27)=46.5, P <0.001), a delay effect (F(2,54)=4.36, P<0.05) , but no group xdelay interaction (F(4,54)=1.31, P>0.05) . A subsequent Newman-Keuls test confirmed that the group effect reflected the exceptionally poor performance of the Fx group, who differed significantly from the Co and Prh groups (P < 0.01 ), who again, did not differ'from each other.

A final analysis, which compared the total scores of the animals taken over both the acquisition and delay conditions, showed the expected significant Fx group effect (F(2,27) = 75.46, P < 0.001). There were, however, no other group differences.

4. Discussion

While fornix (Fx) lesions produced clear deficits on the three tasks which tax spatial memory, lesions of the perirhinal cortex (Prh) had no disruptive effect. Thus rats with neurotoxic lesions of perirhinal cortex were unimpaired on an automated delayed nonmatching-to- position task, a position discrimination with subsequent reversals, and a spatial forced-alternation task. In the object recognition test, however, the Prh lesions were associated with a selective impairment while fornix lesions had no effect. Taken together, these findings indicate that spatial performance can remain unaffected

by Prh damage, and suggest that the hippocampus can contribute to these tasks independent of the perirhinal cortex. The results of the object recognition test also add further support for an anatomical distinction between systems underlying spatial and nonspatial memory [ 1,17]. In considering the implications of these findings attention will focus on the performance of animals with perirhinal damage as the effects of fornix lesions on these various tasks have already been described [3,10,19].

The first test, delayed nonmatching-to-sample (DNMP) was selected as it is sensitive not only to hippocampal and fornix damage [3,10,12] but also to damage in anatomically related regions [2,4,12,21]. While the fornix lesions produced the expected loss of accuracy [3,10], as well as an increase in bias, there was no evidence that the Prh group was impaired on this task. It may be added that the bias change in the Fx animals was more marked than in some previous studies [2,3] as all three measures (Iy, RI and B') were affected. Related to this were decreases in the latencies of some of the responses. These changes in bias in the Fx animals are, however, consistent with many other reports of increases in perseveration and activity following hippocampal system damage [26,33].

In Experiment 2, the rats were trained on a position (lever) discrimination followed by a series of reversals. Consistent with the D N M P task, the Prh group showed no evidence of an impairment on this task while the Fx rats were impaired on the reversal sessions, i.e., those sessions in which the correct lever was on the opposite side to the previous session. Their normal performance


on the initial lever discrimination (Sessions 1 and 2) agrees with other studies of simple position discriminations following hippocampal damage [26,33,46,52]. Similarly, the deficit on the reversal sessions accords both with the finding that hippocampal damage can disrupt position discriminations when they are counter to the animal's preference [23,26,33], and with the high bias scores in Experiment 1.

In the last spatial experiment, forced-alternation in a T-maze, the animals were more able to use distal (room) cues to guide their choices. Once again, only the Fx group was markedly impaired and their scores were close to chance. This result is consistent with many other studies that have highlighted the sensitivity of allocentric memory tests to hippocampal system damage [32,36,46,62]. In contrast, the perirhinal lesions had no apparent effect on performance, either in acquisition or with retention delays of up to 60s.

It can be seen that although these three experiments assessed spatial memory in different ways, the same pattern of results was found. That is, fornix transection produced a clear impairment while rhinal lesions had no effect. This dissociation clearly shows that the rhinal tissue removed in the current study does not form a vital link in the hippocampal system that sub serves many spatial memory tasks. This result also supports and extends the finding that cytotoxic lesions of the parahippocampal region (entorhinal and perirhinal cortices) do not disrupt spatial alternation in a T-maze [55]. In that study it was, however, discovered that aspiration lesions of the parahippocampal region produce a clear deficit on the same task [55], while electrolytic perirhinal lesions can produce a mild deficit on the Morris water maze [67]. This discrepancy between the effects of cytotoxic and conventional lesions has been reported for other, adjacent areas. Thus, cytotoxic lesions of the entorhinal cortex were also found to spare place navigation [27], as were cytotoxic lesions of the entorhinal and subicular cortices [5]. In contrast, aspiration or electrolytic lesion of the same regions consistently produce marked deficits on a range of spatial tasks [29,47,51,55,52,59].

There are a number of possible explanations for this discrepancy between the effects of conventional and cytotoxic lesion. One possibility is that cytotoxic lesions of the entorhinal and perirhinal cortices spare vital fibres of passage that are normally disrupted by conventional lesions. Another possibility is that conventional parahippocampal lesions often extend too deep, and encroach upon the caudal hippocampus [-30]. In either case it would appear that the hippocampus is not dependent of these cortical regions, a view that runs counter to current proposals [13,60]. A quite different possibility is that cytotoxic lesions are more likely to be subtotal, and so spare performance. In which case, the importance of the parahippocampal region for spatial memory may have

been underestimated. In order to resolve these alterna- tives it will be necessary to compare lesions that are very precisely matched in location and extent.

The third experiment examined the effects of lesions of the fornix and of the perirhinal cortex on an object recognition test based on spontaneous exploratory activity. While this test taxes working memory, it has no explicit reference memory component. Lesions of the fornix were found to have no effect on performance of the standard condition of this task. This lack of effect reinforces previous reports that neither fornix lesions nor medial septal lesions, which denervate the hippocampus from its basal forebrain afferents, disrupt object recognition when assessed in this manner [-17,19]. This conclusion is also consistent with a growing number of studies of rats showing that the recognition of nonspatial stimuli can be left intact following hippocampal lesions or fimbria-fornix transection [ 1,37, 55, 52].

In contrast to the animals with fornix lesions, the Prh group appeared unable to discriminate between familiar and novel objects after a retention delay of 15 min in the standard condition. That is, the Prh rats spent a comparable amount of time exploring each object in the choice phase. These same rats performed normally, however, with the shorter delay of just 1 min. This preliminary result, which provides evidence of a deficit on a nonspatial test of recognition, is consistent with a number of other recent studies. Thus aspiration lesions of the perirhinal-entorhinal cortices appear to disrupt delayed nonmatching for both complex objects [38] and odours [48,61]. Related to this it has been found that disconnecting the temporal lobe from the entorhinal cortex can also reduce preference for novelty [40,41]. Furthermore, single unit recordings have revealed neurons in the rhinal cortex of the rat that appear to signal information about the relative familiarity and/or recency of visual stimuli [71]. These findings clearly echo the important discovery that rhinal lesions produce a very severe delayed nonmatching-to-sample deficit in monkeys [34,39,64] and that single units in the primate perirhinal cortex can signal the relative familiarity and recency of stimuli [7,53,73]. Before concluding that these regions are vital for recognition memory in both the monkey and the rat it will, however, be necessary to confirm the nonmatching deficits using cytotoxic rather than conventional lesions. Related to this, it will also be necessary to discover the robustness of the current recognition deficit.

This need is highlighted by the series of tests examining spontaneous recognition for re-configured objects [ 19]. These modified stimuli were used in response to proposals that the hippocampus is involved in the creation of configural associations [63]. The behaviour of the control rats was found to be consistent with a previous, similar study [ 19] as they reacted to the rearrangement of the complex stimulus after 1 min, but not after a


delay of 15 min. Thus the control rats spent more time with A* in the A* vs. A condition (A* is the re-configuration of the sample A) after a 1 min delay, but not after a 15 min delay. Similarly, the control animals spent an equivalent amount of time exploring A* and B after a delay of 1 min, but after 15 min the novel stimulus (B) was preferred. This would indicate that memory for the constituent items is more persistent than memory for their relative arrangement.

The performance of the tornix lesioned animals in the first configural condition (A vs. A*) was similar to that described in a previous study using configured stimuli [ 19], i.e., like the control rats the Fx animals discriminated after 1 min but not 15 min. In the second condition (A* vs. B) it was found that the Fx animals did not differentiate between the two stimuli after 1 min, and although they spent relatively more time with the novel object (B) after a delay of 15 min this effect was not significant. This latter result, which suggests that the Fx animals were still sensitive to the re-configuration of the sample, reveals a slight difference from both the control animals and from the results of a previous study [19]. In that study the rats with fornix lesions showed a clear preference for the novel object after a 15 min delay, i.e., the re-configuration appeared to have been forgotten. It is not clear why the two studies should show this minor discrepancy, but as different stimuli were used in the two studies it is likely that individual stimulus character- istics influenced the degree to which the re-configured stimulus was retained.

The rats with rhinal lesions performed in a similar manner to the control animals in the first configural condition (A vs. A*). Thus they appeared sensitive to the re-configuration after 1 min, but not after 15 min when both groups failed to discriminate between the two stimuli. In the second condition, however, the Prh rats spent significantly more time exploring the re-configured stimulus (A*) than the new object (B) after t min delay. In this respect they seemed, if anything, overly sensitive to the rearrangement of the sample stimulus. For the 15 rain delay the Prh animals performed like control animals, and spent more time exploring the new object. This latter finding appears to be in contrast with the inability of the same rats to discriminate between new and familiar objects after 15 min in the standard condition. This inconsistency must, at present, weaken the conclusion that cytotoxic rhinal lesions are sufficient to disrupt recognition memory. As a consequence current experiments are underway to determine the reliability of these results and to measure the effects of rhinal lesions over a larger range of retention periods.

In summary, the present results provide evidence of a dissociation between the neural substrates for spatial and nonspatial memory. In particular, they show that much of the perirhinal cortex is not a necessary route

for sensory information guiding spatial memory tasks involving the hippocampus. While that route remains undefined it may be noted that the rostral parts of the lateral entorhinal cortex, which were damaged in many of the Prh animals, also appear to be unnecessary for the performance of the spatial tasks used in the current study [27,55]. The results also provided a clear dissociation between the effects of lesions in the perirhinal cortex and the hippocampal system. This dissociation was, however, principally in the opposite direction to that predicted by current accounts of temporal lobe organisation [ 13,60]. It could be argued that the present dissociation is flawed as the perirhinal lesions were incomplete, unlike those of the fornix. This does not, however, seem a very convincing argument as the Prh lesions resulted in very extensive cell loss in the perirhinal region. It is further weakened by the finding that the Prh animals, unlike the Fx animals, were impaired on one version of the recognition task. This evidence of a double dissociation is clearly not consistent with the concept of a unitary memory system. The present findings also bear a striking similarity to a recent report comparing the effects of fornix and perirhinal lesions in the monkey [25]. In that study monkeys with fornix lesions were impaired on a spatial task, but showed only mild impairments on a test of stimulus recognition. In contrast, the monkeys with perirhinal lesions performed normally on the spatial task, but were very severely impaired on the nonspatial recognition task [25]. Once again, the evidence of a double dissociation between the rhinal and hippocampal regions runs counter to the influential notion that these regions form part of a unitary temporal lobe memory system.

Acknowledgement

This research was supported by the Welcome Trust M/90/3028. The authors wish to acknowledge the assis- tance of P. Hunt, S. Nagle and S. Whiteley.

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