Evidence for the Involvement of the Mammillarv Bodies and Cingulum Bundle in Allocentric Spatial Pricessing by Rats

Transcription

1 European Journal of Neuroscience, Vol. 9, pp , European Neuroscience Association Evidence for the Involvement of the Mammillarv Bodies and Cingulum Bundle in Allocentric Spatial Pricessing by Rats N. Neavel, S. Nagle2 and J. P. Aggleton3 'Division of Psychology, University of Northumbria at Newcastle, UK 2Department of Psychology, University of Durham, Durham, UK 3School of Psychology, University of Wales, Cardiff, PO Box 901, Cardiff CFl 3YG, UK Keywords: cingulate cortex, cingulum bundle, fornix, anterior thalamic nuclei, mammillary bodies, spatial memory Abstract Comparisons were made between the behavioural effects of lesions in three inter-related limbic structures: the mammillary bodies, the fornix and the cingulum bundlekingulate cortex. Cytotoxic lesions of the mammillary nuclei produced a marked deficit on reinforced T-maze alternation, but performance gradually improved with practice. Subsequent tests in a cross-maze and a radial-arm maze showed that the animals with mammillary body lesions failed to use allocentric cues, but were able to perform normally in an egocentric discrimination. Three groups of rats with different patterns of either crossed or unilateral radio frequency lesions of the cingulate region were given the same tasks. The profile of results indicated that disruption of those fibres in the cingulum bundle connecting the anterior thalamic nuclei with the hippocampal/retrohippocampal region was responsible for the observed impairments to T-maze alternation and radial-arm maze performance. There was also evidence that disconnection of frontal connections in the cingulum bundle might affect perseverative behaviour, but not allocentric processing. The results add support to the notion of a functional circuit that involves projections from the hippocampus to the mammillary bodies and anterior thalamic nuclei, and from there back to hippocampall retrohippocampal regions via the cingulum bundle. This circuit appears to be vital for normal allocentric processing. Introduction There is growing evidence from both lesion and electrophysiological studies that the anterior thalamic nuclei, along with the hippocampal formation, comprise part of a system underlying normal spatial learning and memory in the rat (Aggleton and Sahgal, 1993; Sutherland and Hoesing, 1993; Aggleton et al., 1995a; Blair and Sharp, 1995; Taube, 1995). In view of the rich direct and indirect anatomical connections between these two regions, this system may largely be reciprocal (Aggleton and Sahgal, 1993; Sutherland and Hoesing, 1993). A direct prediction arising from this proposal is that the integrity of those structures and tracts relaying information between the hippocampus and the anterior thalamic nuclei will also be vital for normal spatial processing. The present study examined this prediction by comparing the effects of lesions in the two major tracts connecting these regions, the fornix and the cingulum bundle. It also examined the effects of selective lesions in the mammillary bodies. The mammillary bodies are of interest as they receive dense hippocampal inputs via the fornix and, in turn, project heavily upon the anterior thalamic nuclei (Cruce, 1975; Swanson et al., 1987; Shibata, 1992). Even though the anterior thalamic nuclei also receive a direct hippocampal-fornical input (Meibach and Siegel, 1977; Swanson et al., 1987), the mammillary bodies may form the principal functional route by which spatially relevant information passes from the hippocampus to the anterior thalamic nuclei. Support for this view comes from the spatial memory deficits noted after mammillary body damage (Beracochea and Jaffard, 1987; Beracochea et al., 1989; Sutherland and Rodriguez, 1989; Saravis et al., 1990; Sziklas and Petrides, 1993; Aggleton et al., 1995a) and mammillothalamic tract damage (Field et al., 1978; Thomas and Gash, 1985). Most of these studies have, however, used conventional lesion techniques with the associated risk of damage to adjacent tracts. We therefore examined the nature and the magnitude of the spatial deficits observed after cytotoxic damage. Particular attention was given to the distinction between allocentric and egocentric spatial processing as this may help to account for the apparent transience or even lack of deficit found in some studies of mammillary body lesions and spatial memory (Aggleton et al., 1990, 1991). The cingulum bundle is the principal route for projections from the anterior thalamus to the cingulate cortices, the hippocampus and the retrohippocampal regions (Domesick, 1970; Shibata, 1993a, b). As a consequence, if the proposed temporal lobe-anterior thalamic spatial system is reciprocal it would be expected that damage to this tract would produce an array of spatial memory deficits similar to those found after anterior thalamic lesions. Unfortunately, the cingulum bundle is located immediately adjacent to the cingulate cortices, Correspondence to:.i. P. Aggleton, as above Received 19 February 1996, revised 1 July 1996, accepted 6 January 1997

2 942 Spatial memory and limbic lesions making it difficult to distinguish between the effects of tract and cortical damage. We tackled this problem by making bilateral, but asymrnetncal radio frequency lesions of the cingulum bundle. The intention was to cut the tract in both hemispheres, but to minimize the extent of bilateral damage to the same portions of cingulate cortex. This approach was designed to complement a previous study in which cytotoxic lesions of the cingulate cortex were compared with radio frequency lesions of the cingulum bundle and adjacent cingulate cortex (Aggleton et al., ). The cytotoxic lesions failed to affect T-maze alternation while the radio frequency lesions produced a marked deficit (Aggleton et al., 1995b). The present experiment would therefore test whether a combination of tract and cortical damage is necessary to produce spatial deficits or whether tract damage is sufficient. A particular advantage of the current preparation is the ability to make comparisons with control animals with exactly the same set of lesions, but restricted to just one hemsphere. As a consequence, both sets of rats (crossed or unilateral lesions) received similar amounts of unilateral cortical damage, but only the crossed lesion group suffered bilateral damage to the cingulum bundle. The first experiment compared the effects of these various lesions on the acquisition and performance of a T-maze forced alternation task (experiment 1) that is highly sensitive to hippocampal, fornical and anterior thalamic damage (Rawlins and Olton, 1982; Aggleton et al., 1986, 1995a). This was followed by two further spatial tests conducted in a cross-shaped maze. The first tested allocentric memory (experiment 2) while the second tested egocentric memory (experiment 3). The allocentric test was essentially the same as the T-maze alternation task (experiment I), but in half of the trials the test run began from a fourth arm that was directly opposite the usual start arm. Thus a strategy that relied on the alternation of body turns (egocentric) would produce errors, but alternating away from the sample place (allocentric) would still lead to a correct choice. The egocentric task (experiment 3) involved rewarding the rat for turning in a constant direction (right or left) irrespective of starting position. Finally, the rats were trained on a standard version of the eight arm radial-maze (experiment 4), so permitting comparisons with a range of other related limbic lesions that have been assessed on this same task (Olton et al., 1982; Saravis et al., 1990; Aggleton et al., 1996). Of particular interest was the performance of the rats with mammillary body lesions, as a recent study indicated that damage restricted to the mammillary nuclei may not be sufficient to disrupt radial-arm maze performance (Sziklas and Petrides, 1993). Two different crossed cingulum bundle (CCB) lesions were studied. One group of animals received lesions at two different anteriorposterior (AP) levels per hemisphere, making a total of four asymmetrical lesion levels (CCB-4). The intention was to ensure that all anterior thalamic-cingulum bundle outputs were disconnected. The other group received more rostral asymmetrical lesions that only involved one AP level per hemisphere, i.e. two asymmetrical lesions (CCB-2). The rostral placement of the CCB-2 lesions meant that although many prefrontal connections in the cingulum bundle should be disconnected, the caudal projections of the anterior thalamic nuclei in the cingulum bundle (including those to the hippocampus) should be largely spared (Domesick, 1970). Two other groups were prepared with precisely the same coordinates as those in the crossed lesion groups, except that all lesions were placed in the same hemisphere (unilateral cingulum bundle lesions; UCB-2, UCB-4). Materials and methods Subjects The study involved 63 naive male rats of the pigmented DA strain (Bantin and Kingman, Hull, UK). These were divided into eight surgical groups. Throughout the experimental period all animals were individually housed under diurnal conditions (14 h 1ightllO h dark), and all testing occurring at a regular time during the light period. The animals were tested on 5 days a week, and prior to test days they were fed -15 g M I laboratory diet (Special Diet Services, Witham, UK) 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 weighed between 210 and 240 g. All animals had free access to water. Apparatus Experiment I: T-maze forced alternation The floors of the T-maze were 10 cm wide and made of aluminium, and the walls were 19 cm high and made of clear Perspex. The stem and both arms were 78 cm long, and at each end was a food well 4 cm in diameter and 0.75 cm deep. The entire maze was supported by two stands 92 cm high. Lighting was provided by fluorescent lights suspended 92 cm above the apparatus, the luminance light level at the choice point being 320 lux. The start arm contained an aluminium guillotine door, set 33 cm from the end of the arm Experiments 2 and 3: allocentric working memoy and egocentric discrimination All testing was carried out in the same T-maze and test room as that used for experiment 1. The T-maze was, however, modified by the addition of a fourth arm, so forming a cross-shaped maze. This arm matched the start arm and, like it, contained a guillotine door. As a consequence all four arms were of the same dimensions, with a food well in each. A slotted Perspex door made it possible to close the end wall opposite whichever stai-t arm was selected, effectively forming a T-maze. For the egocentric discrimination task both guillotine doors (present in just the two start arms) were removed so that all of the arms were as similar as possible. Movable wooden barriers were used instead of doors. Experiment 4: radial-am maze All testing was carried out in an eight-arm radial maze, the floors of which were made of wood. The apparatus was of standard design, but the arms had clear Perspex walls 24 cm high. A food well, 2 cm in diameter and 0.5 cm deep, was present at the outer end of each arm. The arms were connected to a central circular area, 34 cm in diameter, via a set of eight Perspex guillotine doors, each 12 cm high. Strings attached to the doors made it possible for the experimenter to open the doors either individually or simultaneously. The maze itself was mounted upon a table so that it stood 53 cm off the ground. Testing took place in a different room to that used in the preceding experiments, and illumination was provided by three fluorescent lights 140 cm above the maze. The luminance level in the middle of the room was 440 lux. Procedure Experiment I: T-maze acquisition Testing began at least 2 weeks after surgery. Each animal was given several days of habituation to the T-maze, food pellets being placed in both food wells. The experiment proper then began. At the start of each trial, which consisted of two stages, three food pellets (45 mg; Campden Instruments, Loughborough, UK) were placed in each food well and a wooden block was placed by the choice point of the T-maze to close off one arm. On this sample run, the animal was thus forced to enter the open arm and was allowed to eat the food

3 Spatial memory and limbic lesions 943 there. It was then picked up and confined to the start box for a delay of 15 s before the wooden block was removed. On the subsequent test run the animal was deemed to have chosen when it had placed a hind foot in one of the two arms; no retracing was allowed. If the rat had alternated, i.e. had entered the arm not previously visited on the sample run, it was allowed to eat the reward, and was then returned to its cage. If the other arm was chosen, i.e. the same arm as visited on the sample run, the animal was confined to that arm for -10 s, and then returned to its cage. The rats were tested in groups of four, each rat having one trial (two runs) in turn, so that the intertrial interval was -34 min. Each animal received six trials a day for a total of 15 sessions, thus making 90 trials in all. All rats were then tested for six sessions, during which all test runs were preceded by a double-sample run. During this the rat was forced into one arm, rewarded, and then forced to the other arm, and again rewarded. These two sample runs were separated by a gap of 15 s. After another 15 s the rat began the test run. The procedure was identical to that used in the first 15 sessions, except that the rule was to alternate away from the most recent (the second) of the two sample arms. Finally, the rats received a set of three sessions in which the procedure reverted to that in the initial 15 sessions, i.e. a standard single forced sample run. Experiment 2: allocentric working memory Testing began 4 days after the completion of experiment 1. The procedure again consisted of two parts, the first being identical to that described in experiment 1, i.e. on the sample run the animal was forced into one randomly selected cross arm and allowed to eat the reward. All sample runs began in the same start area as used in experiment 1. The second part (test run) was also similar in that the animal was rewarded for entering the arm not previously chosen on the information run. The critical difference was that now on half of the trials the animal was placed in the start area of the arm opposite to that used on the sample run. As the location of the correct choice arm had not altered, an animal that relied on allocentric cues could still alternate to the correct arm on these trials. In contrast, a rat relying on egocentric cues would be expected to make many more errors on those choice trials that began from the opposite arm. It should be added that a barrier was always placed at the mouth of the start arm not being used, so maintaining the T-maze arrangement. All animals received six trials per day for a total of 10 days. Within each day, three test runs were started from the same arm as that used in the sample run, and three test runs began from the opposite arm. Once again there was an intertrial interval of 34 min between trials. Experiment 3: egocentric discrimination Testing began 7 days after the completion of experiment 2. For this task each animal received 12 trials a day, and unlike the previous experiments these were run consecutively, i.e. one immediately after the other. At the start of each trial the rat was randomly assigned to one of the four arms of the cross-maze and confined at the end by a wooden barrier. The arm opposite the start arm had been blocked, effectively creating a T-maze. One of the side arms was baited with three 45 mg pellets, so that the animal was rewarded for selecting the arm in a given direction (always turn right or always turn left). Following a choice, the rat was placed at the end of another randomly assigned arm, confined for -10 s, and the process was repeated, i.e. the rat was rewarded for turning in the same direction irrespective of which of the four arms it had started from. Care was always taken to appear to bait both of the possible choice arms, so that the movements of the experimenter would not cue the animal. Each animal was tested until it reached a criterion level of 230 correct responses over three consecutive sessions (36 trials). Upon reaching this criterion, the correct body turn was reversed for the next and all subsequent sessions. Thus, if the animal had been rewarded for always turning left, it would now only be rewarded for turning right. Each animal again received 12 trials per day, and testing ceased when the animal reached the 30/36 criterion level. The correct body turn (right or left) for the first discrimination was defined by the behaviour of the animal on the first trial of the very first session. On this trial only, both army were baited and the animal was rewarded for entering either arm. On subsequent sessions, until the reversal, the animal was only rewarded for turning in the same direction as it had turned on this very first trial. Experiment 4: radial-arm maze Testing began -5 days after each rat had completed experiment 3. Retraining involved several habituation sessions during which each animal was confined to the maze for -15 min. During this period all of the guillotine doors were raised and the animal was allowed to explore the maze in which food reward pellets had been scattered in each arm. During the experiment proper all eight arms of the maze were baited with three 45 mg reward pellets. The animal was initially placed in the central platform and all the doors at the entrance to the arms were simultaneously raised. When the animal had made a choice by entering an arm it was allowed to eat the pellets. A choice was defined as the rat moving its back legs across the line of door. The animal was then free to return to the central platform, and all of the doors were raised so that the animal could immediately choose the next arm. The session was terminated when the rat had visited all eight arms, or if it had not completed this task within 10 min, or if it had not made a response for 2 min. The experiment consisted of 12 sessions, one per day. Surgical and histological procedures A total of 63 rats were divided into eight surgical groups. These groups were: surgical control (SHAM, n = ll), fornix (FX, n = 6), mammillary bodies (MB, n = lo), extensive crossed cingulum bundle (CCB-4, n = 9), extensive unilateral cingulum bundle (UCB-4, n = 6), rostral crossed cingulum bundle (CCB-2, n = 8), and rostral unilateral cingulum bundle (UCB-2, n = 9). Four animals received anaesthesia but no further surgery (UCON, n = 4). Each of the 59 surgical animals was deeply anaesthetized by intraperitoneal injection of pentobarbitone sodium (Sagatalj at a dose of 60 mgkg. The animal was then placed iq a stereotaxic headholder (David Kopf Instruments, Tjunga, CA), with the incisor bar set at +5.0 to the horizontal plane and the scalp retracted to expose the skull. A craniotomy was made above the sagittal sinus and the dura was cut to expose the cortex above the target region. On completion of all of the surgeries the skin was sutured and sulphanilamide powder applied. The rats also received an injection (i.p.) of 6 ml physiological saline containing 0.3 ml millophyline (etamiphylline camsylate, BPI, a respiratory stimulant. Mammillary bodies For the animals receiving mammillary body lesions (MB), an injection of 0.45 p1 of 0.12 M NMDA (Sigma) dissolved in phosphate buffer (ph 7.2) was made through a Hamilton syringe into a single site in each hemisphere. The stereotaxic coordinates relative to bregma

4 944 Spatial memory and limbic lesions 1. Extensive crossed cingulum bundle lesion (CCB-4) 3. Rostral crossed cingulum bundle (CCB-2) rostral midline caudal rostral midline caudal 2. Extensive unilateral cingulum bundle (UCB-4) 4. Rostral unilateral cingulum bundle (UCB-2) FIG. 1. Diagrammatic representation of the various cingulum bundle lesions. The diagram represents a plan of the cortex, the unbroken horizontal line depicting the sagittal sinus (midline). The broken lines either side of the midline represent the cingulum bundle. X represents the approximate site of the lesion. were AP -2.2 and height mm. The lateral (LAT) co-ordinate was 0.7 mm from the sagittal sinus. Each injection was made gradually over a 4 min period and the needle was left in situ for a further 4 min before being withdrawn. Cingulum bundle All cingulum bundle lesions used a Radionics TCZ (Radionics, Burlington, MA) electrode (0.3 mm tip length, 0.25 mm diameter) equipped with a thermistor. The probe was lowered vertically into the appropriate site and the tip temperature raised to 75 C for 60 s using an RFG4-A Lesion Maker (Radionics). For the most extensive crossed cingulum bundle surgeries (CCB-4), lesions were made at two AP levels in each hemisphere (Fig. 1). At each level two lesions were made in the same tract. This resulted in a total of eight lesions per animal (Fig. 1). The stereotaxic coordinates (mm) of these lesions relative to ear-bar zero were: AP +7.9, LAT 21.1; AP +5.3, LAT, 21.1; AP + 3.7, LAT kl.0; and AP +1.4, LAT t0.9. The deeper lesion was placed (going anterior to posterior) at -2.1, and -1.9 mm from the top of the cortex; the probe was then raised by 0.3 mm and another lesion was made. The lesions alternated between the left and right hemispheres; in half of the cases the most rostral lesion was in the left hemisphere and vice versa The CCB-4 rats were matched by a set of animals that received exactly the same surgical procedure except that the lesions, set at four different AP levels, were all placed in just one hemisphere (UCB-4). In some cases this was the left, in others the right hemisphere (Fig. 1). The more restricted, rostral crossed cingulum bundle lesions (CCB-2) involved lesions at just one AP level per hemisphere (Fig. l), the lesions in one hemisphere being 1.5 mm in front of those in the other hemisphere. Two lesions were placed at each level, one 0.3 mm lateral to the other. The stereotaxic coordinates of the lesions relative to ear-bar zero were (mm): AP +6.5, LAT 1.1 and 0.8 from the sagittal sinus, and AP + 5.0, LAT 0.8 and 1.1. Both rostral lesions were placed 2.2 mm below the top of the cortex while both caudal lesions were 2.3 mm below the cortex. Half of the animals received the more rostral lesion in the left hemisphere and vice versa For the respective control group (UCB-2), the two sets of lesions were placed in the same hemisphere (Fig. 1). Apart from this, the stereotaxic coordinates were identical to those used in the CCB-2 animals. As in the UCB-4 animals, half of the cases received lesions in the right hemisphere and half in the left. Fornix For the animals receiving fornix lesions (FX), two lesions were made in each hemisphere using the radio frequency probe (72", 60 s). The coordinates of the lesions relative to ear-bar zero were (mm) AP +5.3, LAT 20.7, and AP +5.3, LAT The medial lesion was placed 3.7 mm below the top of the cortex, and the lateral lesion was placed 0.1 mm still deeper. The surgical controls (SHAM) received a craniotomy that overlapped with that required for the FX, MB and cingulum bundle surgeries. The dura was then cut to expose the dorsal cortex. The wound was then repaired as for the other surgeries. On completion of the experiment the animals were killed and perfused intracardially with physiological saline followed by 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. Results Histological analysis Mammillury bodies In all ten MB cases the surgery resulted in a circumscribed lesion of the mammillary bodies. In nearly all cases the NMDA injection

5 Spatial memory and limbic lesions 945 FIG. 2. Diagrammatic reconstructions showing the FX and MB cases with the largest (dark grey) and smallest (solid black) lesions in the respective groups. The coronal sections are modified from Swanson (1992) and the numbers correspond to the anterior-posterior level of the sections. resulted in almost complete loss of neurons in all the various mammillary body nuclei (Figs 2 and 3). Only one case showed any appreciable cellular sparing. but the remaining neurons in this animal appeared disorganized. This same case also displayed sparing of the supramammillary nucleus, but in eight of the remaining nine cases almost all of the cells of the supramammillary nucleus had also been destroyed. All MB animals showed a marked enlargement of the mammillary recess of the third ventricle (Swanson, 1992), presumably as a consequence of the cell loss in the adjacent nuclei (Fig. 3). The hypothalamic nuclei rostral to the mammillary bodies were always spared. Fornix The fimbridfornix was completely severed bilaterally in all of the FX cases (Figs 2 and 4). There was, in addition, damage to the immediately adjacent portions of the dorsal thalamus and the corpus callosurn. In some cases the most ventral portions of the anterior cingulate cortex were involved, and in one case there was significant unilateral damage throughout that part of the cingulate cortex above the fomix. The lesion consistently extended caudally to involve the most rostral portions of the hippocampus. Cingulum bundle The cingulum bundle was consistently severed bilaterally in the CCB- 4 group, but in three of the nine cases the lesion bilaterally invaded a restricted portion of the hippocampal field CAI closest to the bundle. These animals were excluded from the study. In the remaining six cases the lesions were centred in the cingulum bundle, although there was inevitable damage to adjacent cortical and callosal regions (Figs 5 and 6). The most rostral lesion typically began just in front of the genu of the corpus callosum and continued in the bundle to the level of the rostral septum. The contralateral lesion in the tract began at around the same septa1 level and extended posteriorly to the middle of the fornix. A third lesion began at the level of the very rostral hippocampus and continued to the level of the habenula. The most posterior lesion was at the level of the splenium. Although all cases showed some damage in the anterior cingulate and retrosplenial cortical regions, in three cases there was no symmetrical bilateral damage, and in the other three this was restricted to a very small region of anterior cingulate cortex. The anterior thalamic nuclei showed a pronounced, bilateral loss of cells in the anterior ventral and anterior dorsal nuclei (Fig. 7). In four of the cases there was a very limited region of unilateral hippocampal damage in the most rostral and dorsal part of the CA1 field. In four cases there was also unilateral damage in a very restricted portion of prelimbic cortex. The lesions in the corresponding unilateral control group (UCB-4) resulted in extensive damage to the cingulum bundle at a variety of AP levels (Fig. 5). The most rostral lesion was around the genu of the corpus callosum, the middle lesions extended continuously in the cingulum bundle from the mid-level of the septum to the level of the habenula, while the caudal lesion was centred in the cingulum bundle at the level of the splenium. The cingulum bundle was very extensively damaged at all of these levels, the lesions typically extending into relatively restricted regions of anterior cingulate and retrosplenial cortex. The anterior ventral and anterior dorsal thalamic nuclei showed a very marked unilateral loss of cells throughout their extent. In all six cases there was a very restricted region of unilateral direct damage to the head of the hippocampus. The CCB-2 group originally contained eight animals, but one was excluded as it showed bilateral sparing of the medial half of the cingulum bundle. The remaining seven animals all displayed a pair of asymmetrical lesions that completely cut the cingulum bundle in both hemispheres (Fig. 8). The more rostral lesion started behind the genu of the corpus callosum and continued caudally to the mid-septa1 level, while the more caudal lesion began at the level of the septum and continued caudally to the level of the fornix (Fig. 8). Although the fornix was never involved, one animal displayed very restricted unilateral damage to the rostral margin of the hippocampus. All cases showed discrete bilateral damage to the anterior cingulate cortex in both hemispheres, although in three cases this damage never occurred in the same portion of tissue and in the remaining four there was only a very restricted region of bilateral symmetrical damage. In almost every case the anterior thalamic nuclei appeared normal on the side of the more rostral lesion, but there was consistent evidence of cell loss and shrinkage in the ventral half of the anterior ventral nucleus and in the anterior dorsal nucleus in the hemisphere with the more caudal lesion (Fig. 7). The lesions in the corresponding unilateral control group (UCB-2)

6 946 Spatial memory and limbic lesions FIG 3. Photomicrographs of coronal sections (Nissl stain) showing the normal appearance the mammillary bodies (a) and the typical appearance of the mammillary bodies in the MB cases (b). The enlargement of the ventricle (V) in the MB case was frequently observed. The abbreviations show the positions of the medial mammillary nuclei (MM), the lateral mammillary nucleus (LM) and the supramammillary nucleus (SUM). Scale bar. 500 pm. In this MB case (b) there is some sparing of the supramammillary nucleus. were very similar but tended to be larger. All nine animals suffered a complete transection of the cingulum bundle, the lesion extending from the level of the genu of the corpus callosum to the caudal limit of the fornix (Fig. 8). Variable amounts of unilateral damage were found in the adjacent anterior cingulate cortex. The anterior ventral (ventral half) and anterior dorsal thalamic nuclei showed evidence of retrograde degeneration on the side of the lesion. Very restricted damage to the rostral margin of the dorsal hippocampus was observed in one case. Following histological analysis, the surgical groups contained 11 SHAM, ten MB, six FX, seven CCB-2, nine UCB-2, six CCB-4 and six UCB-4 animals. Beha vioural analyses The first analyses compared the scores of the surgical SHAM group with those of the anaesthesia-only cases. Following this, the scores of the two cingulate control groups with unilateral lesions (UCB-2 and UCB-4) were compared. The purpose of these initial analyses was to determine if the treatments had any differential effects and, hence. whether it was appropriate to combine these two sets of cases prior to subsequent comparisons. Compurisons between control group F SHAM versus anaesthesia-only animals. The scores for the 11 SHAM and four anaesthesia-only cases for experiment 1 (T-maze alternation)

7 Spatial memory and limbic lesions 947 FIG. 4. Photomicrograph of a Nissl-stained coronal section showing the extent of fornix damage in the FX cases. The arrows show the regions of minor cellular damage in the most dorsal portions of the anterior thalamus. Scale bar, I mm were blocked into groups of three sessions. No group differences were found in blocks 1-5, blocks 6-7 (double-sample run) or block 8 (all P > 0.1). Similarly, no group differences were found for experiment 2 (cross-maze allocentric task, F < 1) or the radial-arm maze task (experiment 4). In this final experiment the scores were first grouped into four blocks, each of three sessions. There were no group differences for either the total number of arms visited (F1,~3 = 1.44, P > 0.1) or the number of correct responses in the first eight choices (FI,~~ = 3.50, P = 0.085). Although evidence of a group difference was present in experiment 3 (egocentric discrimination and reversal), this reflected the higher number of errors made by the anaesthesia-only group (F,,13 = 5.1, P = 0.042). Furthermore, the inclusion of these animals in the control group did not alter any of the subsequent comparisons with the other groups for this experiment. In view of the lack of difference on three of the four experiments, and the fact that the group difference in experiment 3 arose from the poorer performance by the unoperated group and did not alter the pattern of overall results, we combined these two groups to form a single control group (CONT) of 15 animals. Unilateral cingulate/cingulum groups (UCB-2 versus UCB-4). The six UCB-4 and nine UCB-2 animals were compared over experiments 1-4, using the same set of comparisons as used for the SHAM and anaesthesia-only animals. Not surprisingly, the UCB-4 group, which had far more extensive unilateral lesions, tended to perform worse than the UCB-2 group. Nevertheless, this difference did not reach significance. Thus the group comparisons for blocks 1-5, blocks 6-7 (double-sample run) and block 8 of experiment 1 were respectively: FI,J~ = 3.52, P = 0.08; F1,13 = 1.32; ti3 = Similarly, there was no overall group difference in experiment 2 (Fl,13= 2.84, P > O.l), and a two-way analysis of variance on the error scores from experiment 3 (egocentric discrimination.and reversal) was non- significant (F < 1). Likewise, there was no group difference on the radial-arm maze task (experiment 4) as measured by the total number of arms visited (F < 1) or correct responses in the first eight choices (F1,13 = 3.70, P = 0.077). We therefore decided to combine the animals to form a single cingulate control group (UCB) of 15 animals, although the consequences of their combining were monitored. Experiment I: T-maze forced alternation Acquisition (sessions 1-15). The scores for the six sets of rats were grouped into five blocks, each of three sessions, and then compared (Fig. 9). Analysis of variance revealed highly significant effects of group (F5,53 = 40.52, P < ) and blocks of sessions (F4,** = 20.95, P < O.OOOl), as well as evidence of an interaction between the two factors (F20,212 = 1.71, P = 0.034). A subsequent Newman- Keuls test indicated that the scores of the FX group were significantly lower than each of the five other groups (P < 0.01). The CCB-4 and MB groups did not differ from one another, but both scored lower (P < 0.01) than each of the remaining three groups (CONT, UCB, CCB-2). None of these three groups differed from each other. An analysis of the simple effects using the pooled error term (Winer, 1971) showed that all six groups, with the exception of the FX group, showed an effect of blocks of sessions (P < 0.05). The significance of this effect, which reflected the improvement in performance with training, was greatest in the UCB, CCB-2, CCB-4 and MB groups (all P < 0.002). The group X session interaction arose from the failure of the FX group to improve over the sessions (Fig. 9). Double-sample procedure (sessions 16-21). All rats received six sessions (two blocks) in which two sample trials were given, in order to increase proactive interference (Fig. 9). Analysis of variance showed an effect of group (Fs,53 = 4.97, P < 0.001) but not of block

8 948 Spatial memory and limbic lesions FIG 5. Diagrammatic reconstntctions showing the CCB-4 and UCB-4 cases with the largest (dark grey) and smallest (solid black) lesions in the respective groups. The coronal sections are modified from Swanson (1992) and the numbers correspond to the anterior-posterior level of the sections. The levels selected are those that depict the centre of each of the four lesions, i.e. at their largest extent. (F < 1). A subsequent Newman-Keuls test showed that the FX group scored worse than the MB group (P < 0.05) as well as worse than all the other groups (P < 0.01). There were no other group differences and no interaction. This lack of any further difference may have been due to the presence of floor effects, as the control groups found this condition veiy difficult to master. Standard procedure (sessions 22-24). The rats then received a final series of three sessions (block 8) in which testing reverted to the original acquisition procedure (Fig. 9). A one-way analysis of variance revealed a clear group effect (F5,53 = 14.5, P < ). A Newman- Keuls test indicated that the FX group was impaired relative to all other groups (minimum P < 0.01), and that the MB groups scored less than the remaining four groups (minimum P < 0.01). There were no other group differences. Experiment 2: cross-maze (allocentric) task Each group performed ten sessions, comprising 30 trials when the start arm was the same as that used in the sample run, and 30 trials when the start arm was the opposite arm to that used in the sample run (Fig. 10). On the very first session, the percent correct scores of the CONT group were 91.1% for the same arm and 75.6% for the opposite arm. These scores are similar to the overall scores for all ten sessions (same, 92.7%; opposite, 78.2%), so showing immediate transfer to the allocentric test. The groups were compared in a twoway analysis of variance. This revealed a highly significant type of trial effect (F1.53 = 168.1, P < O.OOOl), a clear group effect (F5.53 = 17.64, P < 0.OOOl) and an interaction between these two factors (F5,53 = 6.63, P = 0.0o01). The type of trial effect arose from the poorer levels of performance when the test trial was started from the opposite arm (Fig. 10). In spite of this, the CONT, UCB and CCB-2 animals were able to perform well above chance on opposite arm trials, while the MB and FX animals were much closer to chance. The scores of the CCB-4 group were intermediate (Fig. 10). Exploration of the group effect with the Newman-Keuls test showed that the scores of the FX group were lower than all five other groups (all P < 0.01), and that the scores of the MB group were significantly poorer than the four remaining groups (all P < 0.01). There were no other group differences. There was, in addition, a highly significant group X type of trial interaction. This principally reflected the scores of the FX group which, because of a floor effect, did not decline markedly when tested from the opposite arm. Removal of the FX group from the overall analysis still, however, left an interaction effect (F4,48 = 2.62, P = 0.046). This effect arose from the scores of the MB group, which showed the steepest decline in scores between the two conditions. Ths was confirmed by the presence of a significant interaction when just the MB and CONT groups were compared (F1,23 = 5.76, P = 0.024). Experiment 3: ct-ass-muze {egocentric) task Comparisons were based on the total number of errors made while learning and then reversing the egocentric discrimination to a standard criterion (Fig. 11). One of the CCB-2 animals made >150 errors on the initial acquisition, and seemed unable to reach the criterion score after a total of 26 sessions (ten more than any other animal). This

9 Spatial memory and limbic lesions 949 Rc. 6. Sequence of Nissl-stained coronal sections showing the four lesion placements in a CCB-4 case. The lesions alternate from hemisphere to hemisphere (a, rostral right; b, mid-left; c, midlposterior right; d, posterior left). The most rostral section (a) is at the level of the genu of the corpus callosum, while the most caudal section (d) is near the splenium and above the hippocampus (H). Scale bar, 0.5 mm.

10 950 Spatial memory and limbic lesions FIG 7. Series of coronal sections (Nissl stain) showing the normal appearance of the anterior thalamic nuclei (top left, top right), the unilateral pattern of degeneration observed in the CCB-2 cases (normal, middle row left; degeneration, middle row right) and the bilateral degeneration in the CCB-4 cases (bottom left, bottom right). The arrows point to the regions of cellular degeneration, which are located in the anterior ventral nucleus (AV) and the anterior dorsal nucleus (AD). The scale bar represents 0.5 nun. AM, anterior medial nucleus; SM, stria medullaris. same animal, which had made more than twice the number of acquisition errors of any other animal, was not tested further and was excluded from the analyses. In addition, one of the FX animals became ill during this experiment and was removed. This left five FX animals for experiments 3 and 4. A two-way analysis of variance revealed a significant group effect (F5.3 = 6.07, P = 0.002), but no group X discrimination interaction (F < 1). A Newman-Keuls test showed that the group effect was due to the poorer overall performance of the CCB-2 group, which differed from all of the other five groups (P < 0.01 in all cases). There were no other group differences. An analysis of the simple effects showed that there was a group difference for both the initial discrimination and the reversal (both P < 0.05), and these both reflected the poor performance of the CCB-2 group. As explained, one member of the CCB-2 group was excluded from these analyses as it was unable learn the initial discrimination. Clearly, its inclusion would have made the CCB-2 group deficit even more significant. Experiment 4: radial arm maze acquisition Each group performed 12 sessions and these were split into four blocks, each comprising three sessions. Comparisons were then made using first the total number of arms entered within a session, and second the total number of correct choices made in the first eight arms visited within a trial (Fig. 12). Totul number of arms required to visit all eight arms. Analysis of variance revealed an extremely significant group difference (F5,52 = 36.2, P < ). A Newman-Keuls test showed that the FX group

11 Spatial memory and limbic lesions 95 1 FIG. 8. Diagrammatic reconstructions showing the CCB-2 and UCB-2 cases with the largest (dark grey) and smallest (solid black) lesions in the respective groups. The coronal sections are modified from Swanson (1992) and the numbers correspond to the anterior-posterior level of the sections. made more arm entries than any other group (P < 0.01), while the MB and CCB-4 groups both made significantly more arm entries than the remaining three groups (all P < 0.01). There were no other group effects. There was, as expected, a large effect of session block (F3,156 = 11.24, P < ) as the animals improved with practice. Number of correct arms in the first eight choices. The pattern of results was similar to that found for total number of arms entered (Fig. 12). There was a highly significant group effect (F5,42 = 35.5, P < ) and a Newman-Keuls test showed that the FX, MB and CCB-4 groups all made fewer correct choices than the other three groups (all P < O.Ul), but unlike the total arms visited measure there were no differences between these three impaired groups. No other group differences were found. Once again, there was a highly significant effect of session block (F3,156 = 11.73, P < O.OOOl), as most groups improved. Discussion The rationale for the present study arose from evidence for the involvement of the anterior thalamic nuclei in allocentric spatial processes. To explore this more fully we compared the effects of selective lesions in a major source of anterior thalamic afferents, the mammillary bodies, and the effects of transecting a major pathway for anterior thalamic efferents, the cingulum bundle. Bilateral damage to both structures produced a similar array of deficits on tasks that tax allocentric memory. These deficits closely resembled those found after anterior thalamic damage (Aggleton et al., 1995a, 1Y96), as well as those observed after fornix lesions. Taken together, these results strongly support the notion of a sequential circuit involving the fornix, mammillary bodies; anterior thalamic nuclei and cingulum bundle that is vital for allocentric spatial processes. Mammillary body lesion effects In experiment 1 the animals with mammillary body lesions displayed pronounced deficits on a spatial alternation task. These deficits were a: !- z w 70-0 K W ' T MAZE ALTERNATION cox u)8 CCB-2 CCB.4 FX ME W C E FIG. 9. T-maze alternation (experiment 1). The graph shows the mean percent correct score of each group on the T-maze alternation task. The 24 sessions are grouped to form eight blocks of three sessions. Performance is divided between the initial acquisition stage (blocks 1-j), the double sample procedure (blocks 6-7) and the return to the acquisition procedure (block 8). not, however, as severe as those associated with fornix transection. This difference in severity is consistent with previous studies using the same task (Aggleton et al., 1990, 1995a). It has also been found that anterior thalamic lesions produce a more marked deficit on T-maze alternation than mammillary body lesions (Aggleton et al., 1995a). While the same direct comparison could not be made in the present study, the performance of the MB animals up to the start of the double-sample procedure could be contrasted with that of rats with cytotoxic anterior thalamic lesions that had been tested in exactly the same apparatus with the same procedure (Aggleton et al., 1996). The difference in overall performance levels (MB, 70.2%; anterior thalamic, 62.3%) again indicates that mammillary body lesions do not mimic the full effect of anterior thalamic lesions. This sparing points to the dual involvement of both direct hippocampal-anterior

12 952 Spatial memory and limbic lesions inn c a tccnl --tm --t CCB-2-40 MB SAME ARM ALLOCENTRIC ALTERNATION OPPOSITE ARM FIG. 10. Allocentric alternation (experiment 2). The overall mean percent correct scores for the ten sessions are divided between trials in which the same start arm was used for the sample run and the test run ( SAME ARM ), and trials in which the arm diametrically opposite was used for the test run ( OPPOSITE ARM ). z P W CCB-2 --D- CCB-4 DISCRIM EGOCENTRIC DISCRIMINATION REVERSAL FIG. 11. Egocentric discrimination (experiment 3). The graph depicts the mean number of errors required to reach the acquisition criterion for the body turn discrimination ( DISCRIM ) and its reversal ( REVERSAL ). c s a 8 I- z Y CCB-4 * I SESSIONS RADIAL ARM MAZE FIG. 12. Radial-arm maze (experiment 4). The graph shows the percentage of correct choices made in the first eight trials. The 12 test sessions are divided into four blocks, each of three sessions. thalamic projections as well as the indirect hippocampal-mamillq body-anterior thalamic projections in the performance of this task. In the second experiment, spatial alternation in a modified crossmaze, the control rats showed immediate, accurate transfer when the test trials were started from the opposite arm. This provides clear evidence that the control rats were not only able to use allocentric cues but had relied on such cues for the previous alternation task. In striking contrast, the mammillary body lesions appeared to selectively disrupt the use of allocentric cues. This is because the MB group performed close to chance when run from the opposite arm, i.e. when allocentric, but not egocentric, cues would still permit accurate responding (Fig. 10). This accords with the effects of mammillary body lesions in the Moms water-maze task (Sutherland and Rodriguez, 1989), a test of allocentric processing. In spite of this, the MB animals were able to perform accurately in those test trials starting from the standard same arm, and had gradually improved with training on the previous T-maze alternation task (Fig. 9). This indicates that the MB animals either required many more trials to learn the initial (start) location and its allocentric relationship with the cross arms, or they had acquired another strategy to solve these spatial tasks. The most likely strategy is the monitoring of body turns, and this is consistent with the normal performance of the MB animals on the egocentric discrimination and reversal (experiment 3). Other possible strategies, such as the use of intramaze cues, e.g. odour trails, can be excluded as animals using such cues could still have alternated when started from the opposite arm. The animals with mammillary body lesions were unable to do this. The normal performance of the MB animals on the egocentric discrimination and reversal not only showed that they were able to use such cues, but ruled out a more general deficit in food-rewarded learning tasks. The test did not, however, examine working memory, and so cannot be regarded as an exact analogue of the previous task (experiment 2). It is also the case that initial performance on the egocentric discrimination is likely to be influenced by transfer effects, as animals failing to master the previous allocentric tasks might be expected to find acquisition of this egocentric task easier. It was for this reason that the egocentric task included a reversal, as well as the initial discrimination. Consistent with other reports, mammillary body lesions markedly impaired radial-arm maze performance (Saravis et al., 1990). It was not, however, possible to distinguish between the contributions of the supramammillary nucleus and the remaining mammillary nuclei (Sziklas and Petrides, 1993), as both regions were involved in most lesions. Nevertheless, the present results do show that lesions restricted to these mammillary nuclei are sufficient to induce a substantial deficit, a finding that has recently been questioned (Sziklas and Petrides, 1993). On one measure, total arms visited, the MB deficit was not as severe as that following fornix damage, but using the number of correct responses in the first eight choices there was no difference in the severity of the FX and MB impairments. In view of the pattern of results in experiments 1-3, it is assumed that the MB lesion deficit in this task reflected a failure to use allocentric cues efficiently. The lack of effect on the usage of other spatial cues (e.g. egocentric) may, however, help to explain the variability in the severity of the spatial memory deficit associated with marnmillary body lesions (Aggleton et al., 1991). It is predicted that features in the apparatus that discourage the use of allocentric cues by normal rats (e.g. high opaque walls, lack of distal cues) will help to minimize the mammillary body contribution, and so diminish any apparent lesion effects.

13 Spatial memory and limbic lesions 953 Cingulum bundle lesion effects It has proved difficult to assess the effects of cingulum bundle damage in a manner that is not confounded by the presence of conjoint cingulate cortex damage. One approach has been to compare the consequences of cingulum bundle lesions (with some inevitable involvement of adjacent cortex) with extensive cytotoxic lesions of the overlying cingulate cortex. One such study strongly indicated that the cingulum bundle, but not the cingulate cortex, was needed for normal T-maze alternation behaviour (Aggleton et al., 1995b). The present study used a different approach to tackle the same problem. Asymmetrical, bilateral lesions of the cingulum bundle were produced (CCB-2, CCB-4) and contrasted with the same lesions, but limited to just one hemisphere (UCB-2, UCB-4). As a consequence, only the crossed lesion groups (CCB-2 and CCB-4) would have bilateral tract damage while the extent of direct, unilateral cingulate cortex damage should be comparable between the CCB and UCB groups. This goal was largely achieved as the bilateral, symmetrical damage to the cingulate cortices proved to be very minor in the CCB cases. Furthermore, when it did occur it was restricted to parts of the anterior cingulate cortices, and bilateral cytotoxic damage to this same region leaves T-maze alternation performance unaffected (Neave et al., 1994; Aggleton et al., 1995b). Although the crossed cingulum bundle lesions would also have resulted in the disconnection of some cingulate and retrosplenial tissue in both hemispheres, the placement of the lesions also means that other cortical connections are very likely to have been spared. For example, the retrosplenial-hippocampal connections rhould have been spared bilaterally in the CCB-2 cases and unilaterally in the CCB-4 cases. The various cingulum bundle lesions produced a consistent profile of performance in two of the tests of spatial working memory (T-maze alternation and the radial arm maze). In both experiments the group with extensive rostrocaudal damage (CCB-4) revealed a marked deficit, while the other groups (UCB and CCB-2) were unimpaired. The magnitude of the CCB-4 deficit in the T-maze alternation task was very similar to that observed after mammillary body damage and, like the MB animals, the CCB-4 animals showed clear evidence of improvement in the spatial alternation task with extended practice. Indeed, by the very last block of T-maze sessions they were performing at the same level as the control cases. Unlike the MB cases, the CCB-4 animals were able to perform reasonably well in the crossmaze allocentric task, and although their scores were lower than those of the CONT group in the opposite arm trials this difference was not significant. It would appear, thereforc, that the cingulum bundle lesions spared some aspects of allocentric processing and that the increased spatial demands of the eight-arm radial maze were required to reinstate the spatial deficit. In this task (experiment 4) performance was once again very similar to that observed after mammillary body damage. In contrast, the CCB-4 group performed at a normal level in the body turn task (experiment 3), suggesting that these animals could have used egocentric strategies to gradually aid alternation performance. The sole use of this strategy is not, however, sufficient to explain the scores in the allocentric cross-maze task. It should also be added that the retrograde degeneration in the anterior thalamic nuclei in the CCB-4 cases is probably not sufficient to account for the T-maze deficits. This is because selective lesions in the anterior dorsal and anterior ventral nuclei (where the degeneration was found in the CCB-4 cases) can spare (Greene and Naranjo, 1986) or produce only very mild (Aggleton et al., 1996) T-maze alternation deficits. Furthermore. cytotoxic lesions of the cingulate cortex can produce similar patterns of anterior thalamic degeneration, but again spare T-maze alternation (Neave et al., 1994; Aggleton et at., 1995b). Although some of the cingulum bundle surgeries also resulted in localized, unilateral damage to adjacent regions of the hippocampus, there are good grounds to believe that this did not affect the pattern of results. First, even though animals in the CCB-4 and UCB groups displayed equivalent hippocampal damage, there were striking differences in the performance levels of these groups for three of the four experiments. At the same time the UCB group did not differ from the CONT group, even though only animals in the former group had any hippocampal damage. It was also the case that the UCB-2 and UCB-4 groups did not differ from one another, even though all of the UCB-4 cases displayed some limited unilateral hippocampal damage while only three of the nine UCB-2 cases had any hippocampal involvement. Indeed, when the UCB cases were divided between those with (n = 9) and without any hippocampal damage (n = 6) there were no group differences (P > 0.05) for the T-maze, crossmaze and radial-arm maze tasks using the standard series of comparisons. Finally, the same comparisons were made between the six CCB-4 cases in the study and the three that were discarded as they had bilateral hippocampal damage. There was no evidence that the cases with bilateral damage were more impaired in any of the experiments. The performance of the CCB-2 group is at first glance harder to explain. These animals, like those in the CCB-4 group, received a bilateral transection of the cingulum bundle and yet failed to show an impairment in the three allocentric tasks. In contrast, the CCB-2 rats were impaired in the egocentric discrimination and reversal. There is, however, a critical difference between the two CCB groups. This arises because many of the projections from the anterior thalamic nuclei to the hippocampal and retrohippocampal regions do not join the cingulum bundle until the level of the fornix (Domesick, 1970; Van Groen and Wyss, 1995). As the lesions in one of the hemispheres in the CCB-2 group were consistently rostra1 to this level, these animals only received a unilateral disconnection of the caudally directed anterior thalamic efferents. In contrast, the CCB-4 group would have received a bilateral disconnection of these projections. To account for the CCB-2 results it is therefore proposed that it is necessary to induce a bilateral disconnection of anterior thalamictemporalhippocampal projections in order to disrupt allocentric spatial processes in a consistent manner. This explanation accords both with the unilateral pattern of thalamic retrograde degeneration in the CCB-2 cases (Fig. 7) and with the similar levels of performance in the UCB and CCB-2 groups. The only impairment shown by the CCB-2 group was in experiment 3, the egocentric discrimination and reversal. This deficit was apparent for both the initial discrimination and the reversal. It is very likely that the initial discrimination was affected by negative transfer effects, as animals were required to switch from an allocentric to an egocentric task. For this reason, the CCB-2 discrimination impairment could reflect the differential ability of rats to shift between classes of cues rather than a selective failure to use egocentric information. In fact, there is evidence that the medial prefrontal regions of the rat are involved in both of these processes (Kesner et al, 1989; Bruin et ul., 1994), and so it may be that the deficit in the CCB-2 group reflected a disconnection of these regions following cingulum bundle transection. At first sight, this explanation should predict that the CCB-4 group would also be impaired in this task, and yet this was not the case. It should, however, be remembered that the CCB-4 animals were far less likely to suffer negative transfer effects at the start of experiment 3, as they were performing the previous allocentric tasks inefficiently and had probably adopted egocentric strategies. It is therefore suggested that the initial discrimination deficit in the CCB-2 animals reflected a difficulty in switching cues (Passingham,

14 954 Spatial memory and limbic lesions 1972; Bruin et al., 1994), and that the CCB-4 animals were unimpaired as no such switch was required. The CCB-2 animals were also impaired in the reversal, and this was a consequence of making an unusually high number of perseverative errors. Thus, in the reversal stage the CCB-2 animals had significantly more sessions in which they only achieved scores of between zero and four (out of twelve) than the CONT group (ti9 = 2.33, P = 0.03). There was, however, no difference in the number of sessions in which the two groups scored either 5-8 or 9-12 correct (both t < 1). The effects of the various cingulum bundle lesions point to a number of general conclusions. It now appears that cingulum bundle damage can impair allocentric processing, and that for some spatial tasks this effect is likely to be independent of direct or indirect cingulate cortex damage (Meunier and Destrade, 1988; Aggleton et al., 199Sb). For this reason, cingulum bundle lesion effects need not be a consequence of disconnecting the cingulate cortices (Aggleton et al., 1995b), although this cannot be ruled out in the present study. The difference between the CCB-2 and CCB-4 groups is of especial interest as both suffered bilateral transection of the cingulum bundle, yet only the CCB-4 group was impaired in the allocentric tasks used in the current study. This indicates that a disconnection of prefrontal fibres (cut bilaterally in both the CCB-2 and CCB-4 groups) is not sufficient to impair these allocentric tasks. This is consistent with the failure of cytotoxic lesions of either the prelimbic or anterior cingulate cortices to disrupt T-maze alternation (Neave et al., 1994; Aggleton et al., 199Sb). Furthermore, the similarities between the effects of damage to the mammillary bodies, the anterior thalamic nuclei, and the cingulum bundle (CCB-4 group) suggest that projections from the mammillary bodies to the anterior thalamic nuclei, and thence in caudal cingulum bundle pathways to hippocampal/retrohippocampal regions, are of critical importance for normal allocentric processing. For T-maze alternation this circuit does not appear to require the cingulate or retrosplenial cortices as cytotoxic damage to these areas has no apparent effect (Neave et al., 1994; Aggleton et al., 1995b). Damage to the caudal cingulum bundle is very likely to disrupt those projections from the anterior thalamicllateral dorsal thalamic nuclei to the retrohippocampal region that are thought to support the presence of head direction cells in the hippocampal forniation (Ranck et al., 1987; Mizumori and Williams, 1993; Blair and Sharp, 1995; Taube, 1995). A loss of such information may therefore account for the T-maze deficit in the CCB-4 group, and may explain why a deficit re-emerged in the radial-arm maze task. This is because a loss of head direction information might be expected to be considerably more disruptive to a task where the animal needs to distinguish eight directions (arms) rather than just two. It is, however, the case that the radial-arm maze was in a different room and so acquisition required the learning of new landmarks. This change might have contributed to the re-appearance of the CCB-4 deficit. These considerations raise the broader issue of whether a loss of head direction infoimation is sufficient to account for the cingulum bundle (CCB-4) deficits. In order to solve this question researchers will need to know more about the range of information conveyed from the anterior thalamic regions to the hippocampal regions. AS part of this it will be valuable to assess the impact of such surgeries on allocentric tasks learnt prior to surgery (Sutherland and Rodriguez, 1989). It may also prove necessary to understand better the contribution of those midline thalamic nuclei (reuniens, parataenial, paraventricular) that also project to the hippocampal region (Wyss et al., 1979), especially as they are often involved in anterior thalamic lesions. Unlike the projections from the anterior thalamic nuclei, many of these midline projections do not join the cingulum bundle (Herkenham, 1978). This might, in turn, explain the difference in severity between the effects of anterior thalamic lesions and cingulum bundle lesions on spatial memory. Acknowledgements This research was supported by a grant rom the Medical Research Council, UK. The authors would like to thank S. Whitely, N. Thomas and M. Davies for their assistance. Abbreviations AP CCB Fx MB UCB References anterior-posterior crossed cingulum bundle lesion fornix lesion mammillary body lesion unilateral cingulum bundle lesion Aggleton, J. P. and Sahgal, A. (1993) The contribution of the anterior thalamic nuclei to anterograde memory. Neuropsychologiu, 31, Aggleton, J. P., Hunt, P. R. and Rawlins, J. N. P. (1986) The effects of hppocampal lesions on spatial and nonspatial tests of working memory. Behuv. Brain Res., 19, Aggleton, J. P., Hunt, P. R. and Shaw, C. (1990) The effects of mammillary body and combined amygdalar-fomix lesions on tests of delayed nonmatching-to-sample in the rat. Behuv. Bruin Res., 40, Aggleton, J. P., Keith, A. B. and Sahgal, A. (1991) Both fornix and anterior thalamic, but not mammillary, lesions disrupt delayed nonmatching-toposition memory in rats. Behav. Brain Res., 44, Aggleton, J. P., Neave, N., Nagle, S. and Hunt. P. R. (1995a) A comparison of the effects of anterior thalamic, mammillary body and fornix lesions on reinforced spatial alternation. Behav. Bruin Rex., 68, Aggleton, J. P., Neave, N., Nagle, S. and Sahgal, A. (1995b) A comparison of the effects of medial prefrontal, cingulate cortex, and cingulum bundle lesions on tests of spatial memory: evidence of a double dissociation between frontal and cingulum bundle contributions. J. Neurosci., 15, 727g7281. Aggleton, J. P., Hunt, P. R., Nagle, S. and Neave, N. (1996) The effects of selective lesions within the anterior thalamic nuclei on spatial memory in the rat. Behav. Brain Res., 81, Beracochea, D. and Jaffard, R. (1987) Impairment of spontaneous alternation behavior in sequential test procedures following mammillary body lesions in mice: evidence for time-dependent interference-related memory deficits. Behav. Neurosci., 101, Beracochea, D. J., Alaoui-Bourraqui, F. and Jaffard, R. (1989) Impairment of memory in a delayed non-matching to place task following mammillary body lesions in mice. Behav. Brain Res., 34, Blair, H. T. and Sharp, P. E. (1995) Anticipatory head direction signals in anterior thalamus: evidence for a thalamocortical circuit that integrates angular head motion to compute head dmction. J. Neumsci., 15, Bruin, J. P. C. de, Sanchez-Santed, F., Heinsbroek, R. P. W., Donker, A. and Postmes, P. (1994) A behavioural analysis of rats with damage to the medial prefrontal cortex using the Morris water maze: evidence for behavioral flexibility, but not for impaired spatial navigation. Bruin Res., 652, Cruce, J. A. F. (1975) An autoradiographic study of the projections of the mammillothalamic tract in the rat. Bruin Res., 85, Domesick, V. B. (1970) The fasciculus cinguli in the rat. Brain Res., 20, Field, T. D., Rosenstock, I., King, E. C. and Greene, E. (1978) Behavioral role of the mammillary efferent system. Bruin Res. Bull., 3, Greene, E. and Naranjo, J. N. (1986) Thalamic role in spatial memory. Behav. Brain Res., 19, Herkenham, M. (1978) The connections of the nucleus reuniens thalami: evidence of a direct thalamo-hippocampal pathway in the rat. J. Comp. Neurol., 177, Kesner, R. P., Farnsworth, G., and DiMattia, B. V. (1989) Double dissociation of egocentric and allocentric space following medial prefrontal and parietal cortex lesions in the rat. Behuv. Neurosci., 103, Meibach, R. C. and Siegel, A. (1977) Thalamic projections of the hippocampal formation: evidence for an alternative pathway involving the internal capsule. Brain Res., 134, 1-12.

15 Spatial memory and limbic lesions 955 Meunier, M. and Destrade, C. (1988) Electrolytic but not ibotenic acid lesions of the posterior cingulate cortex produce transitory facilitation of learning in mice. Bahav Brain Res.. 27, Mizumori, S. J. Y. and Williams, J. D. (1993) Directionally selective mnemonic properties of neurons in the lateral dorsal nucleus of the thalamus of rats. J, Neurosci., 13, Neave, N., Lloyd, S., Sahgal, A. and Aggleton, J. P. (1994) Lack of effect of lesions in the anterior cingulate cortex and retrosplenial cortex on certain tests of spatial memory in the rat. Behav. Brain Res., 65, Olton, D. S., Walker, J. A. and Wolf, W. A. (1982) A disconnection analysis of hippocampal function. Brain Res., 233, Passingham, R. E. (1972) Non-reversal shifts after selective prefmntal ablations in monkeys (Macaca mulutta). Bruin Res., 92, Ranck, 5. B., Muller, R. U. and Taube, J. S. (1987) Head direction cells recorded from post subiculum in freely moving rats. Neuroscience, 22, Suppl. 528p, 177. Rawlins, J. N. P. and Olton, D. S. (1982) The septo-hippocampal system and cognitive mapping. Behav. Brain Res., 5, Saravis, S., Sziklas, V. and Petrides, M. (1990) Memory for places and the region of the mammillary bodies in rats. Eur: J. Neurosci., 2, Shibata, H. (1992) Topographic organization of subcortical projections to the anterior thalamic nuclei in the rat. J. Comp. Neurol., 323, Shibata, H. (1993a) Direct projections from the anterior thalamic nuclei to the retrahippocampal region in the rat. J. Comp. Neurol., 337, Shibata, H. (1993b) Efferent projections from the anterior thalamic nuclei to the cingulate cortex in the rat. J. Comp. Neurol., 330, Sutherland, R. J. and Hoesing, J. M. (1993) Posterior cingulate cortex and spatial memory: a microlimnology analysis. In Vogt, B. A. and Gabriel, M. (eds), Neurohiology of Cingulate Cortex and Limbic Thalamus. Birkhauser, Boston, MA, pp Sutherland, R. J. and Rodriguez, A. J. (1989) The role of the fomidfimbria and some related subcortical structures in place learning and memory. Behav. Brain Res., 32, Swanson, L. W. (1992) Bruin Maps: Structure of the Rat Brain. Elsevier, Amsterdam. Swanson, L. W., Kohler, C. and Bjorklund, A. (1987) The limbic region. 1: The septohippocampal system. In Bjorklund, A., Hokfelt, T. and Swanson, L. W. (eds), The Handbook of Chemical Neuroanatomy. Vol. 5, Integrated Systems of the CNS, Parl 1. Elsevier, Amsterdam, pp, Sziklas, V. and Petrides, M. (1993) Memory impairment following lesions to the mammillary region of the rat. Eur. J. Neurosci., 5, Tauhe, J. S. (1995) Head direction cells recorded in the anterior thalamic nuclei of freely moving rats, J. Neurosci., 15, Thomas, G. J. and Gash, D. M. (1985) Mammillothalamic tracts and representational memory. Behav. Neurosci., 99, Van Groen, T. and Wyss, J. M. (1995) Projections from the anterodorsal and anteroventral nucleus of the thalamus to the limbic cortex in the rat. J. Comp. Neurol., 358, Winer, B. J. (1971) Statistical Principles in Experimental Design, 2nd edn. McGraw-Hill, Tokyo. Wyss, J. M., Swanson, L. W. and Cowan, W. M. (1979) A study of subcortical afferents to the hippocampal formation in the rat. Neuroscience, 4,