The effects of fornix transection and combined fornix transection, mammillary body lesions and hippocampal ablations on object-pair association memory in the rhesus monkey

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Behavioural Brain Research, 3-5 (1989) 85-94 Elsevier BBR 00959

The effects of fornix transection and combined fornix transectioh, mammillary body lesions and hippocampal ablations on object-pair association memory in the rhesus monkey R i c h a r d C. S a u n d e r s a n d L a w r e n c e W e i s k r a n t z Department of Experimental Psychology, Universityof Oxford, Oxford (U.K.) (Received 10 August 1987) (Revised version received 14 February 1989) (Accepted 8 March 1989)

Key words: Memory; Habit; Hippocampus; Monkey

Cynomolgus monkeys were tested on an associative memory task in which they had to remember object-object pairings. Eight animals (4 unoperated monkeys and 4 monkeys with lesions to components ofthe'hippocampal-mammillary'circuit) were trained initially on a conditional object-pair association task that was similar to simple discrimination learning, but which involved presentation of object-pairs instead of single objects. Object-pairs were made up from 4 different objects, each with aft identical copy. Animals had to learn which pairings of the 4 objects were rewarded (e.g. AB or CD) and which pairings were not (e.g. AC or BD). Objects appeared as a member of both rewarded and non-rewarded pairs. After several stages oftraining all monkeys reached a high level of performance making greater than 90~0 correct choices. In a performance test the "monkeys had to discriminate between 3 single objects depending on their past associations with the other objects. Animals had to choose the two objects which in initial training were rewarded as an object pair. Monkeys with the 'hippocampal-mammillary'circuit lesions learned the initial object-pair discriminations at the same rate as control monkeys. In the performance test, however, monkeys with the 'hippocampal-mammillary' circuit lesions performed at chance levels, whereas the control monkeys performed significantly better. The results demonstrate a dissociation between the 'habit' and 'mediational' memory systems for ostensibly the same object-pair associations. They also indicate an important contribution of the 'hippocampal-mammillary' circuit in nonspatial association memory.

INTRODUCTION

The present investigation was undertaken to examine the effects of damage to components of the 'hippocampal-mammilla.ry' system (the hippocampus, subiculum, forhix and mammillary bodies are a well-described neural circuit and have collectively been referred to as the 'hippocampal-mammillary' system) on associative

memory in monkeys. An association memory task was devised to examine two types of 'mnemonic function' for the same object-object association. In addition, the-memory task attempted to exploit the mnemonic demands of the pairedassociate memory tasks used extensively with amnesic patients. It has been demonstrated in amnesic patients and'ih experimental animals that the memory

Correspondence:R.C. Saunders. Present address: Clinical Brain Disorders Branch, NIMH, Neuroscienee Res. Ctr., St. Elizabeths Hospital, Washington, DC 20032, U.S.A.

86 impairment after damage involving the hippocampal formation, the fornix, or the mammillary bodies is highly specii~c 1"6'9"!1-13'16-18'23'24. The distinction between the ability to learn and retain certain types of information but not others has been the focus for an'~ilyzing the memory defect as well as the neural organization of intact memory processes. A contrast has been made between, on the one hand, what has been called 'habit' memory and 'habit formation' (responses to specific situations which become automatic through repetition) and, on the other, a more flexible 'mediational memory'. In amnesic subjects it is proposed that 'mediational memory' is severely impaired while learning an.d retention of'habits' remain relatively normal. A number of investigators have described the amnesic syndrome along similar lines, for example, 'reference memory' vs 'working memory '3'2z, 'procedural' vs 'declarative' memory 3~, 'habit' vs 'memory '~~ 'semantic' vs 'episodic' memory 26 and 'habits' vs 'cognitive mediational memory '32. While it is clear that there are differences among the different memory theories, it is equally clear that they are remarkably similar in that they focus upon the dependence on a particular type of mnemonic processing to account for the amnesic patient's success or failure. A dissociation in the amnesic patients' mnemonic capabilities was noticed as early as 1917 by Wechsler in testing Korsakoff patients. He commented that the patients 'were able to reproduce habitually preformed associations almost as readily as normal individuals, but as these became less of a habitual sort the associations were less readily produced' (p. 122) 33. The distinction so impressed Wechsler that two types of pairedassociates were included in his widely used memory scale 34. More recently, this dissociation of success or failure in 'paired-associate' learning and retention has been demonstrated in amnesic patients of various etiologies 23'27'32'36. It appears that success or failure, ,~vithin the task can be controlled by varying the probability of the free generation of the 'response' word when the 'stimulus' word is given. Amnesic patients, in comparison with control subjects, have much more difficulty in 'paired-associate' learning as the response probability decreases (e.g. rhyming

pairs vs random pairs), that is, their performance decreases when the words presented as a pair do not make up non-random associations as found in rhyming pairs or semantically-related pairs. Most studies of the effects of hippocampal, fornix or mammillary body lesions on associative memory in animals have investigated their ability to learn and remember object-food associations, i.e. object-reinforcement associations 6,9,12,14.15, 20,24,30 While the food reward possesses its own stimulus qualities (texture, color, shape, etc.), which can be used in the forming of associations 7, these qualities may be eclipsed by the salience of the hedonic nature of the reward, and this is probably the case in the most frequently, used paradigms. The paradigm used most often is the learning and retention of visual discriminations or variations thereof (e.g. concurrent learning: win- : stay, lose-shift), all with highly similar memory requirements 7. In most cases the animal is required to associate the same food reward (e.g. peanut) with a number of different stimuli. The effects of hippocampal or fornix transections on such memory tasks, however, have not been consistent. After removal of the hippocampal formation (together with other intended or unintended medial temporal tissue) discrimination learning and retention has been shown both to be impaired (Correll and Scoville4, concurrent learning of several discrimination pairs; Mahut et al.~4, retention of single object discriminations; Moss et al. 2~ concurrent learning; Mahut et al. ~5, concurrent learning; Zola-Morgan and Squire 37, retention of object discriminations and concurrent learning) and unimpaired (Orbach et al. 2~, learning of single visual discriminations; Correll and Scoville4, learning of single discriminations; Zola-Morgan etal. 3s, single discriminations; Malamut et al) 7, concurrent learning). It is not clear from these reports whether the difference between the impairment and normal performance ~s due to the mnemonic differences between the tasks, the behavioral testing methods or surgical variations. The purpose of the present investigation was, first, to assess the effects of lesions of the 'hippocampal-mammillary' system (i.e. fornix transection, mammillary body lesion, hippocampal abla-

87 tion) on associative memory in the monkey, but in this case monkeys had to remember object-object pairings and not just object-reward associations. The second purpose was to assess within animals the same object-object associations under two conditions that differ "in the type of mnemonic required for successful performance. MATERIALS AND METHODS

Sllbjects The subjects were 8 mature male cynomolgus monkeys (Macacafascicularis) ranging in weight from 4.5 to 6.5 kg. Mgnkeys were housed in pairs and were maintained on a diet of Dixon's FDI meal mixed with water and daily supplement of fruit. All 8 monkeys had previous experimental experience. Four monkeys, two control (numbers 109 and 113) and two with combiiaed fornix transection, mammillary body lesions, and hippocampal ablations (numbers 86 and 112) had been tested on the task win-stay, lose-shift in an earlier investigation of association memory (Expt. 2, Gaffan et al.9; Saunders and Gaffan, Expt. 324). On the other 4 animals, two controls had previous experience on various visual-spatial conditional discriminations and simple-visual discrimination learning reversals (for details see Gaffan et al.9). The 8 monkeys comprised two groups, the experimental group (n = 4) with those monkeys who had either fornix transections alone (n = 2) or with a combined hippocampal ablation, fornix transection and mammillary body lesions (n = 2), and the control group, 4 animals without damage to the 'hippocampal-mammillary' system.

Surgery Surgical procedures have been described in detail previously24 and only.a brief description is presented here. All operated monkeys were first sedated with an intramuscu!ar injection of Ketalar (10 mg/kg) and anesthetized intraveneously with Intraval Sodium. For the fornix transection, a large midline craniotomy and a unilateral dura flap exposed the midline. The exposed hemisphere was gently retracted to visualize the corpus callosum with the aid of an operating microscope. A small midline slit was made through the corpus

callosum exposing the descending columns of the fornix. The fornix was then transected with a small metal sucker with cautery. Producing the mammillary body lesions followed a similar method except the lesions were made stereotactically with a thermister-controlled probe 0.8 mm in diameter..First, the monkey was placed in a stereotaxic instrument with the correct alignment confirmed using frontal X-ray radiographs. A bone flap was opened, the dura opened and the hemisphere gently retracted. A small slit in the corpus callosum was made in order to position the probe above the midline, as determined by the middle portion of the massa intermedia. Lateral radiographs showed the probe in relation to the bony landmarks of the skull. It had been previously determined that the position of the mammillary bodies could be reliably predicted from the position of the posterior clinoid process TM. The probe was lowered to a position 2 mm dorsal to the most dorsal tip of the posterior clinoid process and 1.75 mm caudal to the rostral tip of the posterior clinoid process. A bilateral lesion of the mammillary bodies was produced from this single midline placement by raising the temperature of the probe tip to 72.5 o C for 60 s. For the hippocampal abalation a craniotomy exposed the dura over the posterior part of the inferior temporal cortex and the occipitaltemporal border. The inferior temporal region was gently retracted and the hippocampal formation exposed and removed with a small gauge sucker. Most of the tissue of the temporal lobe medial to the occipital-temporal sulcus was removed. The boundaries of the ablation were the ventricular surface laterally and dorsally, and the caudal limit of the amygdaloid complex rostrally. All of the hippocampal formation (the dentate gyrus, ammonic subfields CA1-CA4, and the subiculum) and the presubiculum were removed. The combined hippocampal-fornix-mammillary body lesions were conducted in 3 stages 24 with the animals tested extensively after each surgical stage.

Histology Histological procedures and results have been presented in detail elsewhere25. In all cases the

88 lesions were mainly as intended. The fornix transections were complete with extra-fornical damage limited to the ~lit in the corpus callosum and the ventral lip of the cingulate gyrus (area 24). The mammillary body lesions and the hippocampal ablations in't'he two animals with the combined lesions were complete. Damage to tissue outside the intended areas in both cases was minimal. There was, however, in one case (no. 86) slight unilateral damage to the inferior temporal cortex restricted to the ventral surface (for detailed descriptions and photographs through the lesion sites see Saunders and Gaffan24).

Apparatus The training was carded out in a Wisconsin General Testing Apparatus (WGTA) inside a darkened room. Each compartment of the WGTA was illuminated with a 25-W incandescent bulb. White masking noise was present throughout testing. Monkeys were tested in a transport cage, one side of which allowed access to the test array. A black perspex test tray, 15 cm above the cage floor, contained 5 food wells, 1.5 cm in diameter. A center well was flanked by 4 lateral wells, two on the left and two on the right. On each side the two lateral wells were spaced close together, 5 cm apart, with the more medial well 14 cm from the center well. The center well was fixed but the set of lateral wells could be moved towards or away from the monkey. During the initial stages of training the test tray was always kept in as close as possible to the testing cage. The stimuli used were 4 'junk' objects with identical mates; thus the monkey was presented with two examples ofeach object. If single objects are represented by capital letters, the stimulus pairs to be discriminated can be denoted as AB, AC, CD and BD. Object pairs were randomly assigned to be either rewarded ( + ) or nonrewarded ( - ), so when presented in any trial they were either rewarded with a half peanut under each object, represented as A B + , or not rewarded, AC - . Each object appeared as part of a rewarded pair and an unrewarded pair (AB + , A C - , CD +, BD - ). While the reward value of an object-pair remained constant through testing,

the reward value of an individual object changed depending upon the object it was partnered with in a trial. The stimulus pairs were presented according to the schedules described below. As a control procedure the physical objects making up rewarded pairs (e.g. AB and CD) and those making up the non-rewarded pairs (AC and BD) were changed periodically to ensure the monkeys were not discriminating on the basis of some slight difference in the nominally identical objects.

Procedure All training was carded out postoperatively. Once reliable performance was achieved in displacing random objects from over the food wells and retrieving the half peanut reward, formal testing began. There were 5 stages of testing followed by a final performance test. The first 5 stages followed a similar procedure used in simple discrimination learning. The difference here is that the monkey had to discriminate between object-pairs instead of single objects. Progressive stages were given in order to make learning as easy as possible. Stage 1. During this stage the same rewarded object-pair appeared on every discrimination trial (e.g. AB + ), with either of the two possible unrewarded pairs (AC - or BD - ). For each trial two object-pairs were presented over the lateral wells (e.g. AB + vs D B - ), with the left-right position of the pair irrelevant (e.g. AB + vs AC or A C - vs AB + ). The left-right position of objects within the negative object-pair also varied randomly (BD - or DB - ; AC - or CA - ). The left-right position within the positive pair (AB + ), however, remained constant. Thus there were 8 different discrimination trials possible, presented in a pseudo-random order with each one appearing 4 times in 32 trials. A correct response was recorded when the monkey displaced both positive objects in succession; the animal was not allowed to correct an error. Eighty trials were given daily, separated by 15 s intervals, until a criterion performanc~ of 22 correct responses in 24 trials was achieved. Stage 2. This was the same as Stage 1 except that the objects' left-right position within the rewarded pair now varied (i.e. AB + or BA +).

89 There were 16 different discrimination trials which appeared in a pseudo-random sequence with each trial type occurring twice in 32 trials. Training was continued to a criterion of 29 correct responses in 32 trials. Stage 3. This was the same as Stage 1 but with the other positive object-pair (CD + ) substituted for the original pair (AB + ). The negative objectpairs remained the same. During the first 16 trials only, a correction procedure was used. Criterion performance was 22 correct responses in 24 trials. Stage 4. This was the same as Stage 3 but the objects' left-right position within the rewarded pairs varied (i.e. CD q- or DC + ). Criterion performance was 29 correct responses in 32 trials. Stage 5. This was the final stage of the objectpair discrimination before the performance test. It was a combination of Stages 2 and 4. Either of the positive object-pairs (AB + , B A + "or CD + , DC + ) could appear with either of the two negative pairs ( A C - , C A - or B D - , D B - ). This provided 32 different discrimination trials, occurring in a pseudo-random order with each presented once over 32 trials. Animals were trained to a performance criterion of 29 correct responses in 32 trials. Immediately following criterion the monkeys were given the first half of the 'performance test'. Performance test. The performance test was a different measure of the monkeys' ability to remember the object-object associations learned in stages 1-5. The objects were separated, appearing now as single objects rather than in pairs. For each trial, 3 single objects were presented, one over the center well and the other two covering the far lateral wells. The animal had to choose the two objects which had been rewarded as a pair in stages 1-5. The monkey first displaced the object covering the central well; this object determined which of the two laterally placed objects was rewarded. Object A (or D) was placed over the central well with the far lateral wells retracted out of reach but in clear view, covered by objects B and C. As the center object was displaced the lateral objects were moved forward towards the animal. The lateral object which previously formed a rewarded pair in stages 1-5 with the center object (e.g. B for A, or C for D) was

rewarded, the previously negative pairs (C for A, or B for D) were not. A correct response was the displacement of the center object followed by displacement of its previously rewarded partner. Animals were not allowed to correct errors (which they all tried to do). The object covering the central well, A or D, was determined randomly, as was the left-right position' of the objects, C and D, over the lateral wells. Sixteen trials were given with 15-s intertrial intervals. After a week of no testing, the monkeys were given a retention test of stage 5 only and retrained to a criterion of 15 correct responses in 16 trials. Only two monkeys failed to reach this retraining criterion within the first 16 trials (0 trials to criterion); a control.animal took 24 trials and one with the combined lesions took 8 trials to reach criterion. On completion of the retention test they were retested on the performance test for another 16 trials. Finally, as a control procedure, after completing the performance test, the control animals were tested for 64 trials on a new conditional discrimination, using the same paradigm as the performance test but with new objects and no pretraining. Any difference in the level of performancelon the control test compared with the performance test can be attributable to pretraining on stages 1-5 and not just faster learning of the conditional discriminations.

RESULTS

The trials to criterion for all monkeys at each stage (1-5) of learning the object-pair discrimination task are presented in Figs. 1 and 2. The data were analyzed using the Mann-Whitney U test. 29 For the analysis, the data from stages 1 and 2 were combined, as were those from stages 3 and 4. The analysis revealed no differences between the control and experimental groups at the various stages of learning the object-pair associations (stages 1 and 2, U -- 7, P = 0.44, n 1 = 4, n2 = 4; stages 3 and 4, U = 3, P = 0.10, nl -- 4, n2 = 4; stage 5, U = 6, P = 0.34, nl = 4, n2,= 4). Thus, the monkeys with the fornix transections and the combined lesions learned the discrimination task as readily as the control animals. There were no

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Fig. 1. Trials to criterion in learning stages 1 and 2 (top) and stages 3 and 4 (bottom). C: control monkeys; Fx,FHH,MB" monkeys with fornix transection alone (first two from the left in the group) and with the combined fornix transection, hippocampal ablation and mammillary body lesions. There were no differences between groups in the rate of learning on stages 1 and 2, and stages 3 and 4.

apparent differences between the animals with the different lesions throughout stages 1-5. The results of the performance test, however, were different. The data are shown in Fig. 3. Using the binomial test 29, control animals' performance was shown to be significantly above chance (z = 4.51, P < 0.01); each control monkey showed greater than chance performance. In con-

trast, the experimental group's performance was not different from chance (z--0.62, P = 0.28) even though one monkey's (no. 121) individual performance was significantly ab0ve-chance. Accordingly, the two groups' performance were significantly different (U = 1, P -- 0.03, nl = 4, n2 = 4). All monkeys showed improved performance on day 2 (5-10~o) compared with day 1 in the performance test. There was no difference, however, in the improvement of the experimental group compared with that of the control group (rank sums test: Siegel29). Also, there was no striking difference between the groups with respect to the types of errors. Most animals showed a variety of tactics including object and]or spatial preferences. Performance scores of the control animals on the final 'control' performance test is also shown in Fig. 3. Their performance was not significantly different from chance (z < 1) in contrast with their above chance performance in the previous performance test. This result on the control test demonstrates that the learning of the oBject-pair discriminations in stages 1-5 influenced behaviour in the performance test, if it had not performance should have been at chance as in the control test.

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Fig. 3. Percentage correct responses on the performance test for both groups and on the control test for the control animals. Monkeys with damage to the hippocampal-mammillary circuit did not perform above chance while control animals performed significantly above chance except on the control test where performance was not helped by previous learning of the object-pair associations. There was a significant difference between the two groups' performance in the performance test. DISCUSSION The monkeys with lesions of the 'hippocampalmammillary' system (fornix transection, hippocampal ablation, mammillary body lesions) performed as well as the control animals in learning the first 5 stages, the first conditional discrimination. They were also unimpaired in the retention of the discrimination, even after an intervening session of the 'performance test' and a week interval. These data are consistent with previous findings in both patients and non-human primates in which amnesic subjects show normal learning and retention of simple visual discriminations 5't7'28 and extends this type of finding to include more difficult conditional discriminations with compound stimuli. The results indicate that the mnemonic system, important in formation and retention of these habits, must be intact in the present monkeys with damage to the 'hippo-

campal-mammillary' system. That the experimental monkeys were able to perform as well as control monkeys on stages 1-5 also indicates that perceptual, attentional, motivational or motor factors were not responsible for the deficit in the performance test. The impairment of the monkeys with the 'hippocampal-mammillary' circuit damage in the performance test is unlikely to be due to a generalized deficit in conditional discrimination learning, because the monkeys were able to learn the initial and somewhat difficult conditional discrimination in stages 1-5 as readily as normals. Also, in a previous report it was demonstrated in a number of conditional discrimination tasks that monkeys with fornix transections learned as quickly as control animals 9. Thus conditional learning per se is not impaired in monkeys with 'hippocampalmammillary' system damage. Because their performance of control animals

92 was at chance in the control test, for which their was no prior training with the test objects, their good performance in the 'performance test' indicates that they were able to use the object-object associations learned in stages 1-5 to their benefit. It would seem t h a t h form of associative memory was available in control animals in a manner not available for the monkeys with 'hippocampalmammillary' system lesions. This extra availability of the associative memory may reflect the more flexible 'mediational' memory system which is disrupted by damage to the hippocampal system, given that for animals with the hippocampal system damage the 'habit' type memory is available. A similar dissociation in the associative memory capabilities 01" monkeys with fornix transection has previously been demonstrated 9. Two tasks of one-trial object-reinforcement associative memory were used. In both, the monkeys were required to remember whether stimulus objects had been paired with reward or not. In one task, congruent recall base on a win-stay, lose-shift rule, the monkeys had to choose objects in the retention test which had previously been paired with reward. The memory required in this task is similar to that in simple discrimination learning 7. The other task, incongruent recall or win-shift, lose-stay, was essentially the same, except that the rule governing choice of objects at the retention test was the opposite, that is, the monkeys had to choose the objects not previously paired with reward. In the first test, the task required the 'natural' and evolutionary adaptive use of object-reward associations which as a 'habit' might prove beneficial. The probability of responding to a stimulus object increases if it is differentially associated with reward, and repetition would serve to strengthen this relationship. One presentation of the object-reward association may be sufficient for some level of 'habit' to form and thus contribute to the probability of response on the next trial 17. In win-shift, losestay, however, the memory of object-reinforcement associates has to be used or retrieved according to some arbitrary and less automatic rule. In this task, the memory of the reward association is not used in an automatic way, as in

win-stay, lose-shift. Thus, to perform this task requires more than a habit system; a flexible, 'cognitive' memory system may be necessary. Monkeys with fornix transection were impaired in the win-shift, lose-stay, but not win-stay, loseshift. While such a dissociation between the two 'mnemonic' systems can be demonstrated, what has not been determined is the combined contribution of each system to the learning and retention of a 'habit' or 'memory'. In each case, while the mnemonic requirement of a task (discrimination vs recognition tasks) may prefer using one system more than another, it is probable that in the normal subject both can contribute t9 retention of the 'habit' or 'memory'. This combined contribution might answer the discrepancy of the earlier reports on discrimination learning and retention. That is, in some cases the cognitive memory system is more responsible for performance particulary during initial training; but after initial training or when overtraining occurs (which can happen even if it takes numerous trials to reach criterion), the habit system becomes more responsible. For example, when a visual discrimination is just learned it may be using both the memory and habit systems, but with performance relying more on the cognitive memory system than the habit system; but with overtraining, the habit system becomes the most efficient system for retention. Paired-associate learning has been used extensively with amnesic patients to assess both the memory defect and the residual memory abilities 32'36. While the present task is obviously not strictly analogous to the paired-associate tasks used with patients it does, however, have some similarities with it, and it also has a feature not found in other tasks that have been used with animals. The present task, as in paired-associate learning, requires the subject to make associations between stimuli of the same modality. While reinforcement, i.e. the food reward, is necessary in the task, the monkeys must, however, recall the association of the objects in order to collect the reinforcement. Most previous attempts at testing associative memory in monkeys usually required the animal to remember

93 if a stimulus was simply paired with reward or not. This may be inappropriate because reward is a very salient and readily associated stimulus and therefore associations with it may rely heavily on the habit system and less so on the mediational memory system. In coff(rast, in the present experiment the animals had to discriminate between objects on the basis of their past experience with other objects. The present investigation demonstrates that the memory impairment following lesions to the 'hippocampal-mammil!ary system' is specific and dependent upon the type of 'memory' involved. This task demonstrates for the first time the dissociation of the.two 'mnemonic systems' obstensibly for the same stimulus-stimulus associations. The data provide furtlaer evidence for a distinction between a 'habit' system and a more 'flexible' memory system, and also suggests that the 'hippocampal-mammillary' system is an important neural component for the 'flexible' memory system. Furthermore it provides further information about the role of the 'hippoeampal-mammiIlary' system in a non-spatial associative memory task. The data do not rule out, however, and in fact say little about, the contribution of other medial temporal lobe area structures such as the amygdala and the entorhinal cortex to the 'memory system'. These data may help characterize the contribution to memory of the 'hippocampalmammillary' system within the larger limbicdiencephalic memory circuits. It might be said that without an intact 'hippocampal-mammillary' system, animals (and people) can perform 'reflexly' but not 'reflectively'35. ACKNOWLEDGEMENT

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