MEMORY AND THE REGION OF THE MAMMILLARY BODIES

Progress in Neurobiology Vol. 54, pp. 55 to 70, 1998 # 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0301-0082/98/$19.00

PII: S0301-0082(97)00064-6

MEMORY AND THE REGION OF THE MAMMILLARY BODIES VIVIANE SZIKLAS*% and MICHAEL PETRIDES*$ *Department of Neurology and Neurosurgery, McGill University, 3801 University Street, Montreal, Quebec, Canada H3A 2B4 and $Department of Psychology, McGill University, 1205 Dr. Pen®eld Ave., Montreal, Quebec, Canada (Received 14 July 1997) AbstractÐThe contribution of the mammillary region to several classes of learning and memory has been reviewed. There is considerable evidence that lesions of this region of the brain impair performance on tasks that require memory for locations that an animal has visited, but that the de®cit depends both on the amount of damage within the region and the diculty of the task. Such lesions, however, do not appear to impair performance on a variety of spatial conditional associative learning tasks which require the animal to form an association between a place or a scene and a stimulus embedded within it. In addition, damage to the region of the mammillary bodies does not impair the ability to learn a variety of non-spatial memory tasks. These studies suggest that the mammillary region may play a selective role in certain types of spatial learning and memory. # 1997 Elsevier Science Ltd

CONTENTS 1. Introduction 2. Anatomical connections of the mammillary region 2.1. A€erent projections 2.2. E€erent projections 3. Role of the mammillary region in learning and memory 3.1. Spatial memory 3.1.1. Working memory 3.1.2. Place discrimination learning 3.1.3. Conditional associative learning 3.2. Non-spatial learning and memory 3.2.1. Visual discrimination learning 3.2.2. Recognition memory 4. Conclusions References

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ABBREVIATIONS DNMS H LM MB MB-R ML

Delayed non-matching-to-sample Hippocampus Lateral mammillary nucleus Mammillary body Mammillary body and adjacent region Medial mammillary nucleus, lateral

MM SM OC SEM VTA

Medial mammillary nucleus, medial Supramammillary nucleus Operated control Standard error of the mean Ventral tegmental area

In contrast, Victor et al. (1971) argued that damage to the medial dorsal nucleus of the thalamus may be the cause of the severe memory de®cit observed in these patients. More recently, Mair et al. (1979) and Mayes et al. (1988) have each reported two welldocumented cases of anmesia in which the most marked neuronal loss was found within the mammillary bodies. Single-case reports of signi®cant memory impairments following relatively restricted damage to the mammillary region have also been described by Tanaka et al. (1997) and Loesch et al. (1995). Such ®ndings have emphasized once again the critical role of this area for memory processes.

1. INTRODUCTION The severe amnesia that characterizes the Wernicke± Korsako€ syndrome has frequently been ascribed to damage of the region of the mammillary bodies. Classical neuropathological studies have identi®ed a variety of lesions in the periventricular and periacqueductal grey matter in the brains of patients with Korsako€'s psychosis, but the most consistently reported abnormalities were in the region of the mammillary bodies (see Brierley, 1977, for a review). % Author for correspondence. Fax: 514 398 8540. 55

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The ®nding that the underlying neuropathology of Korsako€'s psychosis appears to involve, at least in part, the mammillary region, raises some questions which remain largely unanswered. First, what is the contribution of the mammillary region to learning and memory? Second, what is the minimum critical lesion necessary to elicit the learning and memory de®cits observed after damage to the mammillary region? Finally, given that there is sucient damage to this region, how selective are the impairments? This article will begin with an overview of the anatomy of the mammillary region to demonstrate its relationship to other neural structures known to be important in mnemonic processing. It should be emphasized that throughout this paper, the term ``mammillary region'' will be used to refer to a region centred around the mammillary complex, but including several adjacent nuclei and ®bre systems. The di€erent components of this region of interest will be outlined and their projections described. Subsequently, the literature pertaining to the behavioural signi®cance of the area will be reviewed and several issues that have remained largely unresolved will be addressed by examining some of the work recently carried out in our laboratory.

2. ANATOMICAL CONNECTIONS OF THE MAMMILLARY REGION The mammillary region is situated at the caudal end of the hypothalamus (see Fig. 1). It encom-

passes a number of cell groups that vary in distinctness. Well-de®ned structures such as the medial, lateral, and posterior nuclei form the mammillary bodies. Rostral to the mammillary bodies, lie the premammillary nuclei which, in the rodent, have been classi®ed into dorsal and ventral regions. Dorsal to these structures, the supramammillary and ventral tegmental areas form some of the more poorly delineated masses within the region (for a detailed summary of the morphology of the cell masses in the mammillary region, see Gurdjian, 1927; Rose, 1939; Veazey et al., 1982a). 2.1. A€erent Projections The principal a€erent ®bre system of the posterior hypothalamus has traditionally been considered to be the fornix, which arises from the hippocampal system, and whose ®bres project mainly to the medial mammillary nuclei (Simpson, 1952; Nauta, 1956; Raisman et al., 1966). Input from the hippocampal system, however, is not limited to these structures. Ino et al. (1988) demonstrated a direct projection from hippocampal ®elds CA1±CA4 to the supramammillary area in the cat. This latter projection has been shown to be reciprocal in nature. Retrograde studies using horseradish peroxidase in the rat and cat have demonstrated the origin of the fornix ®bres to be mainly in the subicular complex (Meibach and Seigal, 1977; Allen and Hopkins, 1989; Shibata, 1989), although labelled cells have also been detected in the hippocampus proper (Irle

Fig. 1. Coronal section through the mammillary region showing the various nuclei in the mammillary bodies and adjacent structures.

Memory and the Region of the Mammillary Bodies

and Markowitsch, 1982a) as well as the entorhinal cortex (Shibata, 1988). Some ®bres from the ventral subiculum have also been demonstrated to project to the ventral premammillary nucleus as well as to a region just adjacent to the medial mammillary nucleus (Canteras and Swanson, 1992a). The mammillary region also receives substantial input from various brainstem nuclei through the mammillary peduncle. Fibres originating in the deep tegmental nucleus terminate in the medial mammillary nuclei; ®bres from the dorsal tegmental nuclei terminate in the medial and lateral mammillary nuclei (Briggs and Kaelber, 1971). Wirtshafter and Stratford (1993) suggested that the dorsal and ventral tegmental nuclei of Gudden project in an organized topographical manner in the mammillary complex; the ventral tegmental nucleus projects primarily to the medial mammillary nucleus and the dorsal tegmental nucleus projects mainly to the lateral aspect of the mammillary complex. In a study using rats, Shibata (1987) demonstrated that ascending ®bres to the medial mammillary nucleus originate from parts of the superior-central nucleus of the tegmental nuclei of Gudden. This latter study also reported that various parts of the periaqueductal grey give rise to ®bres directed toward the lateral mammillary nucleus, as well as the supramammillary and premammillary regions. Following injections of WGA±HRP into the medial mammillary body, retrogradely labelled cells were noted in the venral tegmental nucleus (Allen and Hopkins, 1989). In the rat, a€erent projections to the mammillary area have also been demonstrated from both the septal (Conrad and Pfa€, 1976a,b; Swanson and Cowan, 1976, 1979) and the prefrontal (Wouterlood et al., 1987; Allen and Hopkins, 1989) regions. 2.2. E€erent Projections The main e€erent pathways from the mammillary complex consist of the mammillothalamic and the mammillotegmental tracts. The terminals of the mammillothalamic tract are located in the anterior thalamic nuclei. Autoradiographic tracing studies have demonstrated that both the medial and the lateral mammillary nuclei have speci®c projection sites within the anteromedial, anteroventral, and anterodorsal thalamic nuclei (Cruce, 1975; Watanabe and Kawana, 1980; Poremba et al., 1994). The mammillotegmental tract is a major ®bre bundle directed toward the brainstem. The descending connections of the medial mammillary nuclei have been shown to terminate in the ventral tegmental nucleus (Cruce, 1977; Allen and Hopkins, 1990), as well as parts of the pontine grey (Cruce, 1977; Aas and Brodal, 1988, 1989) and the tegmental reticular nucleus (Cruce, 1977; Allen and Hopkins, 1990). The major projection site of the lateral mammillary nucleus appears to be the dorsal tegmental nucleus (Veazey et al., 1982b; Hayakawa and Zyo, 1990). Some ®bres from the mammillary complex have also been noted to project to the ventral tegmental area A-10 in the cat (see Oades and Halliday, 1987). Recent studies have demonstrated that much of the input to the brainstem and thalamic nuclei originates from the same sites within the mammillary

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region. Using a double-labelling method, Hayakawa and Zyo (1989) and Takeuchi et al. (1985) demonstrated that both the medial and lateral mammillary nuclei contain neurons whose collateral ®bres project to both the anterior thalamus and various brainstem nuclei. The hippocampal region has been shown to receive input from the nuclei of the mammillary bodies (Wyss et al., 1979a; Amaral and Cowan, 1980) and the ventral premammillary nucleus (Canteras et al., 1992), as well as the ventral tegmental area (Dilts and McGinty, 1989; Gasbarri et al., 1994). By far the major source of input to the hippocampal system, however, appears to be from the supramammillary region. Anterograde and retrograde labelling techniques in the rat have identi®ed a signi®cant input from this area to the dentate gyrus (Segal and Landis, 1974; Pasquier and Reinoso-Suarez, 1976; Segal, 1979; Wyss et al., 1979b). In the monkey, there is evidence that this projection terminates in the CA2 ®eld of the hippocampus and the dentate gyrus (Veazey et al., 1982b). An investigation in the rat (Van Groen and Wyss, 1990) indicated that supramammillary projections to the hippocampal system extend beyond the regions described above, documenting a€erent connections to the presubiculum and the parasubiculum. A more recent study by Vertes (1992) demonstrated that the hippocampal projections of the lateral supramammillary area were more dense in comparison with those of the medial aspect of this nucleus. In summary, the region of the mammillary bodies receives a substantial input from the hippocampal formation as well as several brainstem nuclei. The ®nding that the connections of the mammillary complex are reciprocal with both the hippocampal formation and certain brainstem nuclei suggests that this region can modulate neural activity in both the brainstem and the hippocampal system. Brainstem nuclei can be in¯uenced through the mammillotegmental tract. The mammillary region can in¯uence activity in the hippocampal system in two ways. First, activity in the hippocampal formation can be modulated directly by way of supramammillary input. A second, more indirect route, can be traced via the mammillothalamic tract to the anterior thalamic nucleus and from there to the hippocampal formation by way of the cingulate cortex.

3. ROLE OF THE MAMMILLARY REGION IN LEARNING AND MEMORY 3.1. Spatial Memory 3.1.1. Working Memory The close anatomical connections of the mammillary region with the hippocampal formation, a system long recognized as critical to memory processing (Milner, 1970; O'Keefe and Nadel, 1978; Weiskrantz, 1978; Squire and Zola-Morgan, 1991), suggest that this region may play a role in memory function. In fact, there is evidence that damage to this area can result in de®cits in the performance of certain spatial memory tasks that have been shown to be sensitive to lesions of various components of

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the hippocampal system. Included in this class of tasks are those in which the animal is required to remember one or more places it has visited over a given period of time. Signi®cant impairments have been noted in the performance of spatial delayed alternation tasks in rats, cats, and monkeys following variable amounts of damage to the mammillary region (Rosenstock et al., 1977; Field et al., 1978; Irle and Markowitsch, 1982b; Greene and Naranjo, 1986). Similarly, early work in our laboratory showed severe and long-lasting de®cits on a variety of spatial working memory tasks after damage to the region of the mammillary bodies (Saravis et al., 1990). Not all investigations, however, have demonstrated severe de®cits in spatial memory. For example, after damage to the mammillothalamic tract, which disrupts the e€erent projections of the medial mammillary bodies to the anterior thalamic nuclei, de®cits on a trial-unique spatial delayed alternation task were only temporary (Thomas and Gash, 1985). Aggleton et al. (1995) found that cytotoxic lesions of the mammillary bodies in rats impaired the acquisition of a forced alternation task in a T-maze, but that the de®cits were not as severe as those of damage to the fornix or the anterior thalamic nuclei. Methodological di€erences as well as lesion size and location may account for the discrepancy in these ®ndings. First, the de®cits may be a function of the length of the delay between successive responses or trials. For example, Holmes et al. (1983) and Beracochea and Ja€ard (1987), using monkeys and mice, respectively, showed that the performance on delayed alternation tasks following lesions to the mammillary region was not markedly di€erent from that of normal animals until the intratrial delay was signi®cantly increased. De®cits in spatial memory tasks, however, have been observed following damage to the mammillary region even at brief delays (Rosenstock et al., 1977), suggesting that other factors must contribute to the impairments seen in these animals. It is important to consider that damage to the mammillary complex and the immediately surrounding region has been very variable across di€erent studies. This point raises the possibility that the memory impairments could be related either to lesion size or to location and that damage to nuclei or ®bre systems adjacent to the mammillary bodies may also contribute to spatial learning and memory de®cits. To test directly the extent to which the preceding factors may account for the inconsistency across studies, Sziklas and Petrides (1993) investigated the e€ects of variable amounts of damage to the mammillary region and the length of intratrial delays on the radial maze, a spatial memory task that is a good indicator of dysfunction in the hippocampal system (Olton, 1978; Harley, 1979; Olton and Papas, 1979; Becker and Olton, 1981). In this task, the animal is placed on a central platform that has a number of di€erent arms extending from it in a radial fashion (see Fig. 2). A pellet of food is placed at the end of each arm and the animal is allowed to visit them to retrieve the pellets. In performing this task, the animal must remember the arms from which it has already retrieved the reward so that it will not return there. Animals were trained pre-

operatively to retrieve the reward at the end of the arms and were then divided into four groups. One group sustained lesions aimed at the medial mammillary±supramammillary complex (MB±SM), another had more extensive lesions to the mammillary region (MB-R) which damaged the whole of the mammillary complex and the supramammillary nucleus, as well as a variety of adjacent structures including the medial ventral tegmental area and the premammillary nuclei. Examples of the two types of lesions to the mammillary region are shown in Figs 3 and 4. A third group sustained lesions of the dorsal hippocampus (H) and a fourth underwent a control operation (OC). Post-operatively, the animals were tested under two conditions. In the ®rst condition, the rats were not con®ned to the central platform between choices; in the second, they were con®ned for a 15 s period before being allowed to make a choice. As can be seen in Fig. 5, lesions restricted to the mammillary and supramammillary nuclei (MB± SM) were not sucient to impair performance in the radial maze even when the memory demands of the task were increased by the addition of an intratrial delay. On the other hand, the performance of animals with extensive lesions to the mammillary region (MB-R) was severely impaired in comparison with that of both the operated control and the restricted MB±SM groups, but was not signi®cantly di€erent from the performance of hippocampal animals in either the no-delay or delay phases of the experiment. These ®ndings indicate that extensive damage to the mammillary region may be necessary to impair performance of the radial arm maze and suggest that damage to structures adjacent to the medial mammillary bodies contributes to the de®cit

Fig. 2. Illustration of the radial arm maze.

Memory and the Region of the Mammillary Bodies

observed in these animals. The latter point is important because it suggests that the extended hippocampal system may require the additional input provided by structures such as the ventral tegmental area, the lateral mammillary, or the premammillary nuclei (Segal and Landis, 1974; Wyss et al., 1979b; Amaral and Cowan, 1980; Canteras and Swanson, 1992b; Canteras et al., 1992) to sustain normal function on this spatial working memory task. This study also showed that another factor determining whether an impairment will be observed in the radial maze task after damage to the mammillary region is the extent to which memory for places is taxed by the addition of delays between di€erent choices. In an earlier study using the radial-maze, Jarrard et al. (1984) found that the performance of

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rats with damage to the region of the mammillary bodies was not signi®cantly di€erent from that of a normal control group. In that study, however, no intratrial delays were imposed between successive choices. Note that in the Sziklas and Petrides (1993) study, the performance of the MB-R rats was signi®cantly above the level of chance during the no delay phase, but performance deteriorated to the level of chance with the addition of a 15 s delay (see Fig. 5). At this point, it is important to mention that although the preceding study emphasizes the role of the mammillary region in spatial learning, it does not address whether the de®cits are speci®cally related to damage of the nuclear masses within the area itself, or whether they are due to disruption of

Fig. 3. Typical lesions to the medial mammillary±supramammillary region (MB±SM).

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passing ®bres damaged by electrolytic lesions. To examine this issue, we injected the neurotoxin Nmethyl-D-aspartate (NMDA) to destroy selectively cell bodies within the mammillary complex and adjacent structures and tested performance in the radial maze under the conditions described above (unpublished observations). As can be seen in Fig. 6, such lesions resulted in a pro®le similar to that observed following lesions created by electrolytic current. A recent study by Neave et al. (1997) also found a signi®cant impairment on performance in the radial maze following cytotoxic lesions restricted to the mammillary nuclei, including the medial, lateral, and supramammillary nuclei, but sparing hypothalamic regions rostral to the mammillary complex. The results reviewed above point to the importance of nuclear masses within this region for at least some types of spatial memory. Furthermore, they suggest that lesions limited to the mammillary and

supramammillary nuclei, provided they damage this area entirely (e.g. Neave et al., 1997), are sucient to disrupt performance. Earlier, it was demonstrated that animals with lesions limited to the mammillary±supramammillary area (MB±SM) were not impaired in a spatial memory task with the intratrial delay used. To investigate whether an impairment might be observed after lesions to the MB±SM with long delays, Sziklas and Petrides (1993) trained animals with damage to the MB±SM on a spatial delayed non-matching-tosample task in the radial maze. On each trial, the animals were allowed to visit only one arm and, after a delay period (15 s), they were allowed to select between the arm they had already visited and one other arm that now contained the reward. Subsequently, they were tested on the same task with di€erent delays (15, 30, 60, and 120 s). As can be seen in Fig. 7 (top), animals with lesions centred

Fig. 4. Typical lesions to the mammillary region and adjacent structures (MB-R).

Memory and the Region of the Mammillary Bodies

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Fig. 5. Mean number of correct responses made during the two test conditions on the radial maze task. Vertical bars indicate the SEM and the dotted line indicates the level of chance. OC, control operation; MB±SM, mammillary± supramammillary area lesion; MB-R, mammillary body region lesion; H, hippocampal lesion.

on the MB±SM were slower to acquire the delayed non-matching-to-sample task in comparison with the operated control animals. Having acquired the task, however, these animals were able to maintain a level of performance comparable with that of the control rats, except at the longest delay (120 s), as shown in Fig. 7 (bottom). These data are consistent with experiments using monkeys (Holmes et al., 1983) and mice (Beracochea and Ja€ard, 1987) in which the de®cits on spatial delayed alternation tasks following damage to the mammillary region were found to correlate with increasing delays. In our own earlier investigation (Saravis et al., 1990), large lesions that invaded not only the MB±SM

Fig. 6. Mean number of correct responses made during the two conditions on the radial maze task. OC, control operation; MB-R, NMDA-induced lesion to the mammillary region.

Fig. 7. (Top) Mean number of trials to reach criterion during acquisition of the delayed non-matching-to-sample task on the radial maze. (Bottom) Mean percentage of correct responses made by each group on the delay conditions of the delayed non-matching-to-sample task. Vertical bars indicate the SEM. The dotted line represents the level of chance; *p < 0.05.

Fig. 8. Mean latency to ®nd the hidden platform for the three groups of animals on the water maze task.

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area, but also several adjacent structures impaired performance on the spatial delayed non-matchingto-sample task with delays of 15, 30, and 60 s. Taken together, these ®ndings argue strongly that the mammillary region is a critical component in a hippocampal circuit underlying spatial working memory, but that the e€ect on memory of lesions to this region depend on the extent of damage and the level of task diculty as measured by the length of time between successive trials. 3.1.2. Place Discrimination Learning The preceding section demonstrated that damage of the mammillary region impairs the ability to remember locations visited within a given session of trials. The question arises whether mammillary damage would impair the ability to learn to go to the same place on every trial. Thompson (1981)

examined the e€ects of damage to the mammillary bodies in rats on spatial discrimination and three successive reversal problems in a T-maze as well as the ability to retain each session's correct location over extended delays. Although the mammillary group made more errors in learning, they were not signi®cantly worse in comparison with control animals on retention trials. These data are consistent with those of Krazem et al. (1995) which showed an impairment in acquisition of a reversal learning set in a T-maze after neurotoxic lesions of the medial mammillary bodies in mice. Aggleton and Mishkin (1985) tested spatial discrimination learning and successive reversals in monkeys with lesions of the mammillary-body region. Because no control animals were available for comparison, their study was restricted to a correlational analysis which revealed a positive correlation between the total

Fig. 9. Diagram of T-maze apparatus used for the visual±spatial conditional associative learning task. The arrows indicate the location of reward for each of the two di€erent stimuli.

Memory and the Region of the Mammillary Bodies

number of trials needed to learn the reversals and the extent of damage in the mammillary region. More recently, Neave et al. (1997) examined the performance of animals with cytotoxic lesions of the mammillary±supramammillary area on an egocentric discrimination task in a cross maze. In that task, the rat was required to make a particular body turn (e.g. always turn right), irrespective of the arm (location) of the maze from which it began each trial. Lesions to the mammillary±supramammillary nuclei did not disrupt performance on this task. The study by Neave and co-workers explicitly tested the ability to learn kinaesthetically distinct responses (i.e. body turns) by making other spatial cues irrelevant. The demonstration that a kinaesthetic solution was possible after mammillary lesions is important because it provides a probable interpretation why the de®cits observed on standard spatial discrimination tasks have been somewhat mild. In learning these tasks, the animals may have used information from body turns and external landmarks to guide their choices. In fact, it is important to report that animals with mammillary damage will use egocentric cues even when correct responses are dependent on external spatial stimuli (Neave et al., 1997, Experiment 2). Two studies used navigational water maze tasks, which are considered to be sensitive to the e€ects of damage to the hippocampal system (Morris et al., 1982), to examine the extent to which the mammillary area is involved in place learning. In the water maze, a pool is ®lled with opaque water and a platform is submerged in a particular location. The animal is placed into the pool, at randomly chosen point and allowed to swim in order to locate the hidden platform using a variety of extra-maze cues. Sutherland and Rodriguez (1989) found that rats with lesions of the mammillary±supramammillary complex had only modest impairments in learning to ®nd the hidden platform in a water maze task. Sziklas et al. (1995) found that extensive lesions of the mammillary region, including the mammillary complex and the supramammillary nucleus, as well as adjacent structures, impaired learning on the water maze task as severely as lesions of the dorsal hippocampus (Fig. 8). Once again, such ®ndings point to the involvement of neural structures adjacent to the mammillary complex in at least some forms of spatial learning.

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which it is more generally involved in spatial learning remains unknown. In a recent series of experiments, we have begun to address this issue by examining the e€ects of extensive damage to the region of the mammillary bodies on spatial conditional associative learning. In spatial conditional tasks, the animal is required to form an association between particular places and arbitrary stimuli (e.g. the animal must learn to choose place X if stimulus A is shown, and to choose place Y if stimulus B is shown). It is important to emphasize a critical di€erence between the conditional associative learning task and the radial maze task described in the section on working memory. In the radial maze, the rat must be able to remember, in a given daily session, the arms from which it has already retrieved the reward so that it will not return there. Performance is thus dependent on the immediately preceding choices in the daily session. In the conditional tasks, on the other hand, the animal must form associations between particular places and stimuli and therefore its response, on any given trial, is based on long-term learning and is not dependent solely on choices made in the daily testing session. Thus, while both types of tasks contain a spatial component, the learning requirements of each are quite di€erent (see also Petrides, 1996). The ®rst of a series of conditional associative learning studies we carried out was conducted in a T-maze as follows. The rat was placed in the start box in the stem of the maze. One of two stimuli was placed at the choice point between the left and right arms (see Fig. 9). The animal learned to enter the left arm upon presentation of one stimulus and to enter the right arm when the other stimulus was presented. Removal of the entire mammillary complex, together with large parts of adjacent neural structures (see Fig. 4 for comparable lesions), did not impair performance on this visual±spatial conditional task (Fig. 10). In contrast, the performance

3.1.3. Conditional Associative Learning The data reviewed above demonstrated that when an animal is required to remember locations, damage to the mammillary region leads to a severe and long-lasting impairment. Milder impairments are observed when the lesion is restricted to the medial mammillary and supramammillary nuclei, sparing adjacent areas. The close anatomical connections of the mammillary region with the hippocampal complex suggest that it is an integral part of the hippocampal circuit underlying spatial memory. However, because only a limited range of tasks (e.g. place learning, working memory) have been used to assess mnemonic processing in animals with damage to the mammillary region, the extent to

Fig. 10. Mean number of days to criterion for each of the three groups on the spatial±conditional associative learning task.

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of animals with damage to the dorsal portion of the hippocampus was signi®cantly impaired in comparison with that of both the operated control group and the mammillary group (Sziklas et al., 1996). The latter results are consistent with other reports in the literature documenting de®cits in conditional associative learning after hippocampal damage in both humans (Petrides, 1985) and monkeys (Ga€an and Harrison, 1989). The striking dissociation between the e€ects of lesions to the hippocampal and mammillary region on spatial conditional learning suggests that the role of the mammillary area in spatial learning may be limited to certain tasks. In a subsequent study, the animals were required to learn the following rule. If the stimulus was located close to the stem of a T-maze, the reward was always found in the right goal arm. If the same stimulus was placed at the wall opposite the start arm, however, the reward was located in the left

arm of the maze (Fig. 11). It was hypothesized that the formation of associations between spatial stimuli and spatial responses would be a more stringent test of this type of spatial conditional associative learning. Consistent with the results of the preceding conditional task, the performance of animals with lesions to the mammillary region was not di€erent from that of the control animals (Fig. 12, unpublished observations). Why were animals with mammillary lesions not impaired on the spatial conditional associative learning tasks, even though such animals are impaired in learning other spatial tasks, such as the water maze? It is possible that the animals were able to solve the conditional tasks by using kinaesthetic cues rather than extra-maze spatial cues. That is, animals may have learned to associate visual cues with motor responses, such as turning left or right, rather than to associate the visual cues with distinct allocentric lo-

Fig. 11. Illustration of the apparatus used for the spatial±spatial conditional associative learning task. The arrows indicate the location of the reward when the stimulus is placed in each of the two possible positions.

Memory and the Region of the Mammillary Bodies

Fig. 12. Mean percentage of correct responses made by each group over blocks of learning trials on the spatial± spatial conditional associative learning task and on the probe trials.

cations. This issue was examined directly over two probe sessions in the spatial±spatial conditional task discussed above by rotating the maze 180 degrees, but leaving the reward in the same allocentric position. Thus, while in the original condition, a left turn would yield a reward if the stimulus cue was placed at the far end of the maze, in the rotated condition; a right turn would be the correct response. As indicated on the right of Fig. 12, the performance of both operated control and mammillary groups deteriorated, suggesting that all animals were relying on visual±motor (i.e. kinaesthetic) associations to solve the task. The possibility thus remained that animals with mammillary lesions may be impaired in a conditional learning task in which a kinaesthetic solution is not possible. This question was explored in the following experiment. Animals were trained on a spatial±visual conditional associative learning task in which they were required to learn associations between arbitrary stimuli and external spatial locations (Sziklas et al., 1996). On each trial, two visual cues were placed side by side in either the north or the south end of an open ®eld located in a room with a variety of extra-maze stimuli (Fig. 13). On any given trial, the position of the two stimuli in the north or south end of the ®eld, as well as their relative left or right pos-

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Fig. 14. Mean number of days to criterion for each of the three groups on the spatial±visual conditional associative task; *p < 0.05.

ition, was determined according to a random schedule. The animals had to learn to approach one cue when the cues were at the north end of the ®eld and to approach the other cue when the cues were located at the south end. It is clear that in this test situation, the problem could not be solved on the basis of pure kinaesthetic response strategies. Consistent with the results of the preceding conditional experiments, we found that extensive lesions to the region of the mammillary bodies did not a€ect the acquisition of this task. In contrast, the performance of animals with lesions of the dorsal hippocampus was severely impaired, remaining near the level of chance throughout testing (Fig. 14). Taken together, the data from the conditional associative learning tasks suggest that the mammillary region does not play a critical role in forming associations between spatial cues, whether egocentric or allocentric, and visual stimuli. 3.2. Non-spatial Learning and Memory 3.2.1. Visual Discrimination Learning Several studies have examined the role of the mammillary region in the acquisition of visual discrimination learning. In these tasks, the animals are

Fig. 13. Diagram of the open ®eld used for the spatial±visual conditional associative task.

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faced repeatedly with the same set of visual stimuli and are only rewarded when they select certain of these stimuli. Thus, animals gradually learn to select the ``positive'' stimuli because such responses are reinforced, and to stop responding to the ``negative'' stimuli that are never reinforced. There is considerable evidence that animals with extensive lesions of the amygdalo-hippocampal region can learn such simple visual discriminations (Zola-Morgan et al., 1982; Malamut et al., 1984). Consistent with these ®ndings, a number of studies have demonstrated that extensive lesions of the mammillary region do not lead to impairments on the acquisition or reversal of simple visual discriminations (Thompson and Hawkins, 1961; Holmes et al., 1983; Saravis et al., 1990).

3.2.2. Recognition Memory The discrimination task demonstrates that the mammillary region is not critical for linking a nonspatial visual stimulus to reward. A di€erent issue is the extent to which damage of the mammillary region may a€ect the ability to recognize whether or not a non-spatial stimulus has been previously seen. Tests of object recognition require the animal to discriminate between familiar and novel stimuli on the basis of one trial. On a delayed non-matching-tosample task, the animal is faced with an object during the sample phase. The object is then hidden from sight for a certain period of time and, subsequently, the same stimulus and a novel one are shown simultaneously, the animal being rewarded for choosing the novel one. Trial-unique tests of recognition use a completely new pair of stimuli on each trial. Aggleton and Mishkin (1985) found only a small impairment in relearning a preoperatively acquired delayed non-matching-to-sample task with 10 s intratrial delays in monkeys with variable amounts of mammillary and extra-mammillary damage. Furthermore, the performance of experimental animals was not signi®cantly di€erent from that of a control group when longer delays or list lengths of objects were subsequently used. ZolaMorgan et al. (1989) tested the post-operative acquisition of such a recognition task in monkeys with variable amounts of damage within the mammillary complex. The results showed impairments in learning the basic task, as well as poor performance with longer intratrial delays. When the task was administered again 18 months later, no signi®cant di€erences were noted between the experimental animals and a normal control group. Although these data suggest that long-lasting global memory de®cits do not occur after lesions to this region, they do not address the issue whether more taxing non-spatial memory tasks, such as those that require judgements based on relative recency of recurring stimuli, might be impaired by such damage. In contrast to trialunique recognition tasks, tests of relative recency use a ®nite pool of objects or stimuli throughout testing. Thus, to choose correctly on the choice phase of each trial, the animal must remember which of several equally familiar stimuli it has seen most recently.

Aggleton et al. (1990) found no impairments after cytotoxic lesions to the mammillary bodies in the rat on a delayed non-matching-to-sample task in which the same sample stimuli were used every ®fth session. These authors also suggested that the same rats remained unimpaired when the stimuli were repeated within a single test session. We examined the performance of animals with extensive lesions of the mammillary region on a recognition task in which two stimuli recurred throughout testing (Sziklas and Petrides, 1993). The task was run on a modi®ed ``plus'' maze (see Fig. 15) as follows. A rat was placed in the central compartment and one of the three doors was raised. The animal was allowed to run to the end of the arm and consume the reward. The sample arm was either black or white. When the rat had returned to the centre, the door was lowered and approximately 2 s later, the doors to the other two arms (the choice arms) were raised. One of these arms was the same as the one seen in the sample phase, the other was di€erent. The rat was rewarded only if it chose the arm that was di€erent form the previously presented stimulus arm. The location and colour of the sample and choice arms were varied randomly for each trial but the two stimulus arms recurred on every trial. The

Fig. 15. Diagram of the apparatus used for the non-spatial recognition task.

Memory and the Region of the Mammillary Bodies

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Fig. 16. (Left) Mean percentage of correct responses made by each group during the acquisition phase of the non-spatial DNMS task. (Right) Mean percentage of correct responses made in the delay conditions of the DNMS task by the mammillary body region (MB-R) group and those animals in the operated control (OC) group which were able to achieve criterion during the acquisition phase. The dotted line indicates the level of chance; the ®lled circles represent individual animals.

rat was unable to use extra-maze spatial cues to solve the task because the top of the entire maze was covered with an opaque cloth and lit from within. As indicated in Fig. 16 (left), animals with lesions of the mammillary region were able to acquire this complex non-spatial task. In fact, the performance of these animals was signi®cantly better in comparison with that of an operated control group, suggesting that destruction of the mammillary area may result in a facilitation in the task described. Furthermore, as can be seen in Fig. 16 (right), having acquired the task, the performance of the mammillary animals under conditions of extended delay was not signi®cantly di€erent from

that of normal control animals that had also demonstrated some learning during the acquisition phase of the experiment. In contrast, the performance of the same animals was severely impaired when they were tested on the acquisition and retention of an analogous spatial non-matching-tosample task using only two arms of the radial maze (Fig. 17). This ®nding is important when one considers that the spatial task is much easier to learn, as de®ned by the amount of training necessary for control subjects to reach a similar level of performance on the two tasks. The results of this last study suggest that an animal with lesions to the mammillary region that may

Fig. 17. (Left) Mean percentage of correct responses made by each group in the training trials of the spatial DNMS task. (Right) Mean percentage of correct responses made by each group in the delay conditions of the spatial DNMS task. The dotted line represents the level of chance; vertical bars indicate the SEM.

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have diculty in using spatial response strategies may be able to function more eciently in an environment where such cues are irrelevant. 4. CONCLUSIONS The work summarized above suggests that the region of the mammillary bodies contributes to a neural circuit underlying spatial learning and memory. Signi®cant impairments after damage of this area have been observed on tasks requiring memory for one or more places. The severity of the impairment, however, appears to depend on at least two factors: the level of task diculty and the amount of damage to the region. The extent to which individual nuclei contribute to performance on working memory and place learning remains unknown. These issues will have to be addressed by future studies. Evidence for the selective nature of the impairment following mammillary damage was also presented. Based on the work to date, this region does not appear to be important for the capacity to form associations between arbitrary stimuli and spatial cues. In contrast, it was shown that damage to the hippocampus can result in poor performance on some conditional associative learning tasks. Such dissociations between these two neural systems emphasize the uniqueness of the mammillary contribution to spatial mnemonic processes. With regard to non-spatial learning, the available data suggest that even a signi®cant disruption of the mammillary area need not lead to impairments in visual discrimination and recognition memory. Together, the ®ndings that damage to the mammillary region can lead to signi®cant and selective spatial memory de®cits are consistent with the idea that at least one component of the memory problems in patients with Korsako€'s syndrome may be due to disruption of neural structures in and around the mammillary bodies. REFERENCES Aas, J. and Brodal, P. (1988) Demonstration of topographically organized projections from the hypothalamus to the pontine nuclei: an experimental anatomical study in the cat. J. Comp. Neurol. 268, 313±328. Aas, J. and Brodal, M. (1989) Demonstration of a mamillo-pontocerebellar pathway: a multi-tracer study in the cat. Eur. J. Neurosci. 1, 61±74. Aggleton, J. P., Hunt, P. R. and Shaw, C. (1990) The e€ects of mammillary body and combined amygdalor-fornix lesions on tests of delayed non-matching-to-sample in the rat. Behav. Brain Res. 40, 145±157. Aggleton, J. P. and Mishkin, M. (1985) Mammillary-body lesions and visual recognition in monkeys. Expl Brain Res. 58, 190±197. Aggleton, J. P., Neave, N., Nagle, S. and Hunt, P. R. (1995) A comparison of the e€ects of anterior thalamic, mamillary body and fornix lesions on reinforced spatial alternation. Behav. Brain Res. 68, 91±101. Allen, G. V. and Hopkins, D. A. (1989) Mammillary body in the rat: topography and synaptology of projections from the subicular complex, prefrontal cortex, and midbrain tegmentum. J. Comp. Neurol. 286, 311±336. Allen, G. V. and Hopkins, D. A. (1990) Topography and synaptology of mamillary body projections to the mesencephalon and pons in the rat. J. Comp. Neurol. 301, 214±231.

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