Amygdala, hippocampus and associative memory in rats

ELSEVIER

Behavioural Brain Research 61 (1994) 175-190

BEHAVIOURAL BRAIN RESEARCH

Research Report

Amygdala, hippocampus and associative memory in rats M ~ Angeles Peinado-Manzano* Del~armme,t<> de Psicoh~gia B(~sica. Psicobiologia y Metodologia de &s Ciencias del Comportamie,to. Facultad de Psicoh~ght. Universidad de Sahmtanca, Salamanca. Spa#7

(Received 6 January 1993; revised 25 January 1994: accepted 25 january 1994)

Abstract

Male wistar rats received either electrolytic or sham lesions into the amygdala, hippocampus or amygdala plus hippocampus, or were assigned to an unoperated control group. In Experiment 1, all lesioned and control animals were tested for the ability to master an associative memory test in which recall was assessed over delays ranging between 10 and 180 s. The goal of Experiment 2 was to study the susceptibility to proactive interference following the above mentioned types of damage. The role of the amygdala and hippocampus in remembering stimulus-magnitude of reward associations was evaluated in Experiment 3. Lesions of the dorsal and ventral hippocampus had no effect on acquisition of the associative memory test, but disrupted the animals' performance in the task after 120 and 180 s delays. The same lesions increased the sensitivity to interference but did not impair the performance of several stimulus/ magnitude of reward discriminations. By contrast, amygdala lesions impaired the acquisition of the associative memory paradigm and the animals' performance over the successive delays. Moreover, the animals with these lesions were not able to learn the stimulus/ magnitude of reward discriminations, although they did not show an increased susceptibility to interference. Combined damage to the amygdala plus hippocampus severely disrupted the acquisition of the associative memory paradigm and the animals" performance over successive delays. The same damage increased the susceptibility of the animals to interference and impaired the performance in the stimulus-magnitude of reward discriminations. Key words: Amygdala; Hippocampus: Associative memory; Interference; Magnitude of reward; Rat

I.

Introduction

Two medial temporal lobe structures, the amygdala and the hippocampus appear to be involved in learning and memory processes. The results of numerous experiments unequivocally point to the involvement of the hippocampus in these processes but do not concur as regards the role played by it in monkeys [7,13,58,71,72] and rats [3,23,26,31,36,37,41,48,53,55,62,70]. The contribution of the amygdala to certain kinds of learning and memory is also supported by abundant experimental evidence in several species, such as rats [5,6,11,21,22,33-35,45-49] and monkeys [25,38,60]. However, some studies have reported that amygdala lesions do not disrupt the performance of certain learning and memory tasks in rats [2,3,6,9,52] and monkeys [73 ]. The selective contribution of each of these

For all correspondence. Address: C, Velazquez, n <, 6. Salamanca, Spain.

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structures to the different kinds of learning and memory processes constitutes a major problem in current research. The goal of the present study was to gain further insight into the role of the amygdala and the hippocampus in memory of the association between sensorial stimuli and reward. The amygdala has been proposed to be strongly involved in the formation of stimulus-reward associations. Amygdala connections with cortical areas in several species [42,64] and the electrophysiological characteristics of amygdala neurons in monkeys [40] support such a proposition. However, behavioural data concerning this role are not consistent. Damage to the amygdala has been found to impair the performance of tasks requiring stimulusreward association in rats [ 12,27,28,45-49] and monkeys [ 15,17,25,39,58 ]. Additionally, behavioural data indicate that the amygdala appears to be part of the circuitry responsible for the association of auditory stimuli with an affective meaning [32]. By contrast, some studies have reported no deficits or the occurrence of inconsistent effects in the performance of tasks involving a stimulus-

0166-432894 S7.00 5) 1994 Elscvicr Science Publishers B.V. All rights reserved SSDI 0 1 6 6 - 4 3 2 8 ( 9 4 ) 0 0 0 0 7 - 3

176

M.A. Peinado-Manzam) / Behavioural Brain Research 61 ;1994 ~ 1 7 5 - 1 9 ~

reward association following damage to the amygdala in monkeys [ 1,4,8,16,74] and rats [20,28]. Ealier studies did not find consensus as to the role played by the hippocampus in memory of the sensorial stimulus-reward association. Hippocampus damage has been reported to disrupt the performance of tasks which require this association, such as discrimination learning, acquisition of learning sets and concurrent discrimination, in monkeys [72] and rats [10,63]. By contrast, some studies with monkeys suggest that it does not play a major role in this kind of memory [51,54] although it does appear to be critical for the formation of object-place associations [44]. In a previous work, I found that partial lesions of the dorsal hippocampus impaired the acquisition and retention of place-reward associations but had no effect on the acquisition and retention of sensorial stimuli-reward associations in rats [48]. This work attempts to determine whether the amygdala and the hippocampus afford different contributions to memory of the association between a sensorial stimulus configuration and reward. Experiment 1 investigated the effects of separated or combined lesions of both structures on the performance of a task which requires the formation and retention of a visuo-tactile configuration-reward association after different delays. The main objective of this experiment was to evaluate the hypothesis that the hippocampus and the amygdala contribute to this kind of associative memory through differential involvement in the acquisition and consolidation of the sensorial stimulusreward association. Experiment 2 examined the susceptibility to proactive interference that follows the abovementioned types of damage. The differential role of the amygdala and hippocampus in processing the magnitude of reinforcement was investigated in Experiment 3.

2. Experiment 1 2. I. Materials' and methods" Subjects" Forty-three male Wistar rats, between 60 and 80 days old and initially weighing between 270 and 300 g were individually housed in cages containing a constant supply of food and water and maintained on a 12-h light-dark cycle (light period from 08.00 to 20.00 h) in a temperaturecontrolled room. Apparatus and materials Behavioural testing was carried out in a T-maze. A guillotine door opened from the start box (31 x 20 x 19 cm) into the stem alley (31 x 20 x 19 cm). Entry to the goal boxes (31 x 20 x 19 cm) was through one-way swing doors (arm alleys, 31 x 20 x 19cm; choice area, 25 x 25 × 19 cm).

The floor of the alleys and the choice area was made o{ stainless steel wires. Each alle} was illuminated wilh tx~ 10-W white light bulbs located under the ceiling of the arm alleys and 21 cm above the floor. No other ~ illumination was present during experimental sessions. During the associative memory testing, animals ~ere trained with a set of 50 visuo-tactile configurations differing according to three variables: ~ hite/black pattern, shape and texture. These stimulus configurations were presented over the arms of the maze. l'he floor and walls of the arm alleys (from the choice area to 1_he goal-box) were lined with various materials, such as cardboard, sandpaper, wire gauze, stainless steel, wood, cloth, scourer, plastic or stone painted or engraved in differeni, black and,or white pa~terns.

General procedure The animals were randomly assigned to seven groups: an unoperated control (unop), n = 4; 3 sham-operated (hippocampus, H.S; amygdala, A.S; amygdala plus hippocampus, A + H.S), n = 5 for each group and 3 lesioned (hippocampus, H.E; amygdala, A.E; amygdala plus hippocampus, A + H.E), n = 8 for each group. All animals were handled for 5-7 rain once a day before surgery and postoperatively for 5 days before the behavioural training started. Surgery Approximately 1 week after arrival, the rats of tile H.S, A.S, A + H.S, H.E, A.E and A-~ H.E groups were subjected to stereotaxic surgery. The animals were deprived of food and water 18 h prior to surgery. The rats were anaesthetized with an intraperitoneat injection of equithesin (20 mg/kg) and subsequently placed in a DKI900 stereotaxic device with the upper incisor bar set at + 5 mm above the horizontal zero plane. Bilateral electrolytic lesions were made by passing a t.5-mA current fi.w 18 s through a stainless steel electrode (0.4 mm in diameter~ insulated except for 0.5 mm at the tip. Stereotaxic coordinates of the lesion sites, according to the system B ol Pellegrino et al. [50] atlas, were the following: (1) Site A. R-C: -2.2 mm; M-L: 1.5 turn and D-V: 3.0 ram; site B. R-C: -2.2 ram; M-L: 2.5 turn and D-V: 3.0 ram; site C R-C: -3.2 ram; M-L: 5.5 mm and D-V: 5.0 mm and sit~ D, R-C: -3.2 ram; M-L: 5.0 mm and D-V: 6.5 mm (H.I~ group); (2) site A, R-C: 0.0 ram; M-L: 4.6 mm and D-V 7.4 mm; site B, R-C: -0.8 ram; M-L: 5.4 mm and D-V 7.5 ram; site C, R-C: -0.8 ram; M-L: 3.5 mm and D-V 8.7 mm and site D, R-C: -0.8 mm; M-L: 4,5 mm ant D-V: 8.6 mm (A.E group); (3) both (1) and (2) sets ~ coordinates (A + H.E group). The sham-operated group, received the same surgical procedure except that the lesio~ electrode was not inserted ip.to the amygdala or hippo

M.A. Peinado-Manzano / Behavioural Brain Research 61 (1994) 175-190

campus but immediately above them and no current was passed. The above coordinates were used but with D-V: (1') Site A, 2.0 mm; site B, 2.2 ram; site C, 2.0 mm and site D, 1.8 mm (H.S group); (2') site A, 6.5 mm; site B, 6.5 mm; site C, 7.0 mm and site D, 5.2 mm (A.S group); (3') 1' + 2 ' ( A + H . S group). Bistraciclin 500 (0.2 ml) was injected intramuscularly every 6 h over the 3 days following the operation.

Behavioural training The rats were gradually food deprived to 85 o; ad libiturn body weight for one week before training began and maintained at this weight level throughout the experiment. After a T-maze exploration phase, the animals of each group were trained on an associative memory basic task. Twenty-four h later, associative memory was tested through progressively longer delays. T-maze exploration. The rat was placed in the start box and 10 s later the door was raised and the animal was allowed into the alleys and goal boxes for a daily 15-min period. One standard 45 mg pellet of food was placed in each goal box. When the rat entered a goal box and ate in less than 90 s this exploratory phase was considered to be finished. Associative m e m o r y testing. The animals were tested for their ability to remember which of two equally familiar visuo-tactile configurations had been previously paired with a food reward. Each trial consisted of two phases, acquisition and test, with a 10-s interval between each. In the acquisition phase two - positive and negative - novel visuo-tactile configurations were presented successively, each one twice. The sequence of the stimulus configuration presentation varied equally between two, ABAB and ABBA, combinations with a 10-s interval between each presentation. The animals could get a food reward in both presentations of one (positive) of the two configurations, determined at random. In each presentation, by obstructing one arm with a square of cardboard, the rat was forced to enter the other whose floor and walls were arranged with the corresponding configuration. The stimulus configuration presentations were assigned to the right and left arms semirandomly; each stimulus was presented once in each arm. The rat was placed in the start box and the door was open immediately, allowing the animal to run to the choice point, where the cardboard square obliged it to choose the open arm. In the rewarded stimulus configuration, the animals were given a standard 45-mg pellet of food. Fifteen s after the rat had entered the goal-box, it was placed back in the start box and confined there until the next presentation. In the test phase, the same visuotactile configurations were simuhaneously presented in the right and left arms at random, and the rat could enter either arm (but only one). Food, a standard 405-mg pel-

177

let, was located in the goal box of the arm arranged with the configuration rewarded in the acquisition phase. Once it had entered the arm of its choice, the animal was not allowed to leave. In each trial the animal had to remember in the test phase which stimulus had been rewarded during the acquisition phase of that trial. A daily training session consisting of 5 trials with a 60-s intertrial interval was given 5 days a week to each animal until it had reached the learning criterion. This consisted of reaching a score of at least 80°,, correct responses in the choice period during 4 consecutive daily training sessions plus 1000,~, correct responses in one more consecutive daily training session up to a maximum of 750 trials. I distinguished four learning criterion levels on the animals' performance throughout the training sessions. The first level consisted of reaching 80"o correct responses in the choice period in a daily training session; the second level implied the attainment of 801~o correct responses during two consecutive daily training sessions; the third level involved the attainment of 80 °o correct responses during 4 consecutive daily training sessions, and the fourth level implied the attainment of the complete learning criterion: 80~'o correct responses during 4 consecutive daily training sessions plus 100 ° b correct responses during one more consecutive daily training session. Each trial used a new pair of stimulus configurations taken from a pool of 50 configurations. Thus, each stimulus configuration appeared only once every 25 trials, on average, on every fourth day of training. Twenty-four h after the attainment of the learning criterion, the animals were tested for their ability to remember the visuo-tactile configuration-reward associations for longer than 10 s. The animals were trained on the same task but with successively longer delays of 60, 120 and 180 s between the acquisition and test phases in successive blocks of 50 trials at each delay condition. The animals received a daily training session consisting of 5 trials with a 60-s intertrial interval for 10 days at each delay condition.

Histological ver(lication At the completion of all the behavioural experiments each rat was injected with an overdose of equithesin (40 mg/kg) and perfused intracardially with an 0.9°5 saline solution followed by a 10°; formalin-saline solution. The brain was removed and stored for 30 days in a 30!~o sucrose/10 o~, formalin solution. Frozen sections, 40 Fern thick, were prepared and each section was mounted and stained with Cresyl violet for microscopic examination of the locus of the lesion and its extent. These were determined by an observer unaware of which group the animals were from. Each section in the lesioned area was magnified, projected and traced by

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M . A . Peinado-Manzano / Behavioural BIzliu Research 6 ] ~1994i 175- ] 9#

means of a microscope connected to a computer. The areas showing damage were located and determined by microscopic examination. These data were outlined on the tracings and were then transferred to drawings of matching normal brain sections taken from the atlas of Pellegrino at al, [ 50]. All hippocampus-lesioned animals showed bilateral damage of the hippocampus (Fig. 1). In all cases, the dorsal hippocampus was damaged, the area between 1.2 and 3.2 mm posterior to the bregma point was primarily afected. In addition, all animals presented damage to the ventral hippocampus, primarily affecting an area between 2.8 and 4.0 mm posterior to the bregma point. Of the 8 rats given bilateral lesions to the amygdala, 7 showed bilateral damage, including most of the lateral, basolateral, basomedial, medial, cortical and central nuclei (Fig. 2). Structures adjacent to the amygdala were not

-1.2~

involved. The other rat. which sustained additional bilateral damage to the piriform cortex, was excluded from the statistical analyses and from the final results. The combined damage to thc amygdala plus hippocampus included most of the dorsal hippocampus, the ventral hippocampus and most of the amygdata (Figs. 3A and B). Of the 8 animals given bilateral combined lesions, 7 showed bilateral damage to the dorsal and ventral hippocampus, primarily affecting at~ area betwecn 1.2 and 3,6 mm and 2.8 and 4.0 mm posterior to the bregma point, respectively. These animals also presented bilateral damage to the amygdala including the lateral, basolateral, basomedial, medial, cortical and central nuclei. The other rat was excluded from the statistical analyses and from the final results because its amygdaloid damage was unilateral and it also sustained additional bilateral damage to the thalamus.

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Fig. 1. Representation of histologically verified hippocampus bilateral lesions in successive brain sections taken front the atlas of Petlegrino et ai. [50] The lined area represents the maximum extent of the damage. The dark area shows the minmmm extent of the Lesi~m mal! rals

.,11.A. Peinado-Man--am~ / Behavioural Bra#7 Research 6l (l 994j 175-190

179

O.B

0.4

o.o

-0.4

Fig. 2. Representation of histologically verified amygdala bilateral lesions. Conventions as in Fig. 1.

Statistical anahwes A two-factor analysis of variance (ANOVA) with repeated measures on the last factor was employed for Group x Learning criterion level design for the analysis of the differences between groups in terms of the number of trials required to reach the learning criterion levels on the basic task. The score of an animal for each level was the total number of trials it had received when it reached that level. When an animal did not reach a learning criterion level after a maximum of 750 trials, its score at that level was 750. Another ANOVA with repeated measures on the last factor was employed for Group x Delay design for the analysis of the differences between groups in term of the percentage of correct responses given during the last 50 training trials on the basic task and the 50 training trials at the successive delays. One-factor analyses were used in the analysis of the differences between groups at each delay separately and in the analysis of performance throughout the different delays for each group separately. Where necessary, the Newman-Keuls test and two-tailed t-test were used after the ANOVAs. 2.2. Results The performance of the groups during the associative memory testing on the basic task and when longer delays were imposed is summarized in Table 1 and Figs. 4 and 5. The Group × Learning criterion level ANOVA indicated that the groups did not improve equally during the training on the basic task. It revealed a group effect, k~,34 = 14.42, P<0.001, a learning criterion level effect, F3.~c~ = 125.69, P<0.001, and a non-significant interaction among groups and learning criterion levels, Fls '

me = 1.21, P > 0.25. More detailed comparisons are given below. Control groups. The unoperated control and shamoperated, H.S, A.S and A + H.S, groups showed the same performance pattern during training on the basic task (see Table 1 and Fig. 4). Statistical analyses confirmed that they did not differ significantly in their level of performance (maximum t34 = 0 . 5 , P > 0.1 for each two group comparison). Hippocampal lesions. Statistical analyses confirmed that hippocampus-lesioned animals exhibited a similar performance to that of the control groups throughout the training on the basic task (mean number of trials required to reach the learning criterion: H.E = 393.1; Unop = 360; H.S = 383; H.E vs. Unop, I34 = 0.37, P > 0.1; H.E vs. H.S, t34 = 0.11, P > 0 . ) . (See Table 1 and Fig. 4). Amygdala lesions. All the amygdala-lesioned rats exhibited severe deficits in the acquisition of the learning criterion in the basic task with respect to the control (mean number of trials required to reach the learning criterion: A . E = 750: U n o p = 360; A.S = 337; A.E vs. Unop, t34=4.34, P < 0 . 0 1 ; A.E vs. A.S, t34=4.6, P < 0 . 0 1 ) and the hippocampus-lesioned (mean number of trials required to reach the learning criterion: H.E = 393.125, t~4 = 3.97, P < 0 . 0 1 ) groups. They needed more training sessions to reach the first 3 levels of learning than the control and hippocampus-lesioned animals and did not reach the fourth level (see Table 1 and Fig. 4). They were able to reach a level of 80°° correct responses in a daily training session for a long time before they were able to maintain this level over 2, 3 and 4 consecutive days. However, these animals were unable to reach 100" o correct responses in a single day.

180

M.A

Peinado-Manzano

Beha vioural Brain Research 6 / i,'/()94) 175-19~

A

_1.2

I-2o8

t

I

-1.6

-2,0

_2.4

t

t4,,0

,0

Fig. 3. A: representation of histologically verified hippocampal bilateral damage in the amygdala plus hippocampus-iesioned group. Conventions as in Fig. 1. B: representation of histologically verified amygdaloid bilateral damage in the amygdala plus hippocampus-lesi~ned group. Conventions as in Fig. I.

M..4. Peinado-Manzapm ,' Beha vioural Brain Research 61 (l 994) 175-190

Tablc 1 Mean ( _+S.E.M.) number of trials required to reach the first (L 1), second (L21, third (L31 and fourth (L4)learning criteria levels in the associative memory basic task

Group

kl

L2

L3

L4

Unop S.op H.E A.E A+H.E

175.00 +_45.41 124.66+ 19.22 158.75+_36.83 465.00 _+37.84 425.71 _+82.55

245.00_+51.76 184.00+16,90 258.75+_30.43 560.00_+39.12 500.71 +_84.32

278.75.+38.26 261.66_+ 17.80 306.87_+21.60 712.14+18.28 613.57+76.52

360.00+35.76 366.00_+23.64 393.12+33,28 750.00_+0 645.71 _+67.53

Unop, unoperated control group; S.op, sham-operated groups: H.E, hippocampus-lesioned group; A.E, am~gdala-lesioned group: A + E.E, amygdala plus hippocampus-lesioned group.

Combhwd leskms. The rats with combined lesions of the amygdala plus hippocampus were severely impaired throughout the training with respect to the control (mean number of trials required to reach the learning criterion: A + H.E =645.714; U n o p = 360; A + H.S = 378; A + H.E vs. Unop, t 3 4 = 3 . 1 8 , P<0.01; A + H . E vs. A + H . S , t34- 2.98, P < 0.01) and the hippocampus-lesioned (mean number of trials required to reach the learning criterion: H. E = 393.125, t34 - 2.81, P < 0.01) groups. Interestingly, two animals showed a similar performance to that of the control and hippocampus-lesioned groups and reached the complete learning criterion. These animals showed less severe damage to the amygdala. The other animals exhibited a similar performance to that of the amygdala-lesioned group; they needed more training sessions to reach the

181

first 3 levels of learning than the control and hippocampuslesioned animals and did not reach the fourth level (see Table l and Fig. 4). Furthermore, they were able to reach a level of 80 % correct responses in a daily training session for a long time before they were able to maintain this level over 2, 3 and 4 consecutive days. However, these animals were unable to reach 100°o correct responses in a single day. The progressive increase in the delay between the acquisition and test phases progressively impaired the performance of all groups. The Group x Delay ANOVA revealed a group effect, F6,34 = 8.81, P < 0.001, reflecting the poor performance of the lesioned groups, P < 0.05 with the N e w m a n - K e u l s test; a delay effect, F3,m:= 130.92, P<0.001, and a significant interaction between groups and delays, Fls, lo: = 1.80, P < 0.05, thus reflecting the different effects of the lesions on the different delays. More detailed comparisons are given below. Control groups. Statistical analyses confirmed that the unoperated control and sham-operated, H.S, A.S and A + H.S, groups did not differ significantly in their levels of accuracy throughout training at the 60 s delay (maximum 1 3 4 = 0 . 9 0 , P > 0.1 for each two group comparison), 120 s (maximum t34 = 1.65, P > 0.1 for each two group comparison) and 180 s (maximum t34= 1.09, P > 0 . 1 for each two group comparison) delay (see Fig. 5). All of them were progressively impaired as the delay between the acquisition and test phases was increased, although the animals maintained a level of performance above chance in the 180 s delay.

800 • • A x •

o

GOD

100 -

Unop S.op H.E A.E A+H.E

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C

400

L

0

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90

O

80

O

L_ L

200

O

70

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t: i

i

i

Unop

S. Op.

H.E

'

A.E

60

'

A+H.E

GROUPS

Fig. 4. Mean number of trials required to reach the complete learning criteria in the associative memory basic task. (Unop, unoperated control group; S.op, sham-operated groups; H. E, Hippocampus-lesioned group; A.E, amygdala-lesioned group: A + H.E. amygdala plus hippocampuslesioned group).

50

i

i

i

i

Basic Task

60 s

120 s

180 s

DELAYS

Fig. 5. Mean percentage of correct responses in the last 50 trials of training on the associative memory basic task and the 511trials of training at the successive dela)s. (Abbreviations as in Fig. 4).

182

M.A. Pe#tado-Manzano Behavioural Brain Research 61 (1994) 175-19#

Hippocampal lesions. The animals with damage to the hippocampus were unimpaired with respect to the control groups on the performance at 60 s delay (mean percentage of correct responses: H . E = 85.25; U n o p = 8 7 ; H . S = 8 4 . 4 ; H . E vs. Unop, t34=0.51, P > 0 . 1 ; H.E vs. H . S , t34 = 0.27, P > 0 . 1 ) . By contrast, a clear impmrment with respect to the unoperated control group became apparent when they were tested at the 120 s delay (mean percentage od correct responses: H.E = 75.5; U n o p = 8(/.5, I34=3.88, P < 0 . 0 1 ) . Their performance at 180 s delay dropped to chance level and was significantly worse than the performance of the control groups (mean percentage of correct responses: H . E = 55.75; U n o p = 75.5; H.S = 72: H . E vs. Unop, I 3 4 = 4 . 5 , P < 0 . 0 1 ; H . E vs. H.S, t34 = 3.64, P < 0 . 0 1 ) . Their level of performance decreased significantly as the 120 and 180 s delays were imposed, F3,~ = 71.3, P < 0.001. Their performance was significantly worse in the 120 (mean ~o of correct responses = 75.5) and 180 s (mean ~o of correct responses = 55.75) delays than their own performance during the last 50 trials of training on the basic task (mean Oo of correct responses = 89, 121 = 6.37, P < 0 . 0 1 , and t~l = 13.57, P < 0 . 0 1 respectively) and at the 60 s delay (mean ','/Oof correct responses = 85.25, t z l = 4.24, P < 0 . 0 1 , and t21 = 11.43, P < 0 . 0 1 respectively). Furthermore, their performance was significantly worse in the 180 s delay than in the 120 s delay (t2~ = 7.20, P < 0.01 ). (See Fig. 5). Before considering the performance deficits shown by the amygdala-lesioned and amygdala plus hippocampuslesioned rats in the different delays, it should be taken into account that these animals had not reached the complete learning criterion in the basic task. Furthermore, their level of accuracy decreased progressively as successive 6{), 120 and 180 delays were imposed. Amygdala lesions. The amygdala lesioned rats showed a worse performance than the control groups in the 60 s (mean percentage of correct responses: A . E = 76; U n o p = 87; A.S = 86.4; A.E vs. Unop, 134 = 3.14, P < 0.01; A.E vs. A.S, 134=2.96, P < 0 . 0 1 ) and 120 s (mean percentage of correct responses: A.E = 59.71; U n o p = 80.5; A . S = 7 8 ; A.E vs. Unop, 134 = 14.64, P < 0 . 0 1 ; A.E vs. A . S , t34 = 12.86, P < 0 . 0 1 ) delays. Moreover, they showed a worse performance than the hippocampus-lesioned group in the 60 s (mean percentage of correct responses: H . E = 85.25, t34 = 2.63, P < 0.05) and 120 s (mean percentage of correct responses: H . E = 75.5, 134 = 10.76, P < 0.01) delays. Their level of performance in the 120 and 180 s delays dropped near to chance level. In the 180 s delay condition, their performance was impaired with respect to the control groups (mean percentage of correct responses: A . E = 5 3 . 7 1 ; U n o p = 7 5 . 5 ; A . S = 7 2 . 4 ; A.E vs. Unop, 134 = 4.93, P < 0.01 ; A.E vs. A.S,/34 = 4.20, P < 0.01). Their level of accuracy decreased significantly when the 120 and

180 s delays were imposed, F3,1~, = 35,89, P < 0 . 0 0 I . Their performance was significantly worse in the 120 (mean ~i, of correct responses = 59.71) and 180 s (mean % of correct responses = 53.71) delays than their own performance during the last 50 trials of training on the basic task (mean ",, of correct r e s p o n s e s = 7 8 . 8 5 , I ~ = 6 . 8 , P < 0 . 0 1 and t~s = 8.77, P < 0 . 0 1 respectively) and at 60 s delay (mean I~,, of correct r e s p o n s e s = 7 6 , I~s=5.68, P < 0 . 0 i and t1~ = 7.65, P < 0 . 0 1 respectively). (See Fig, 5), Combined lesions. The amygdala plus hippocampuslesioned animals were significantly impaired with respect to the control (mean percentage of correct responses: A + H . E = 7 0 . 8 5 ; U n o p = 87; A , H.S = 86.4; A + H.E vs. unop, 134 = 4.29, P < 0 . 0 1 ; A + H . E vs. A + H.S, 13~ = 3.38. P < 0.01) and hippocampus-lesioned (mean percentage of correct responses: H.E = 85.25, ".4 =: 3.77, P < 0.01) groups in the 60 s delay. Additionally. they were also impaired with respect to the control (mean percentage of correct responses: A + H . E = 6 2 . 8 5 ; U n o p = 8 0 . 5 ; A + H.S = 78.4: A + H.E vs. Unop, 134 = 12.37, P < 0 . 0 1 : A ~ H.E vs. A + H . S , l~ 4 = 1 0 . 5 0 , P < 0 . 0 1 ) and hippocampus-lesioned (mean percentage of correcl responses: H , E = 7 5 . 5 , t~4 = 8.49, P < 0 . 0 1 ) g r o u p s in the 120 s delay. Their performance dropped to chance level for the 180 s delay and was significantly worse than the performance of the control groups (mean percentage of correct responses: A-~ H . E = 56.28; U n o p = 75.5: .~X+ H.S = 71.2; A + H.E vs. Unop, I34=4.38, P < 0 . 0 1 ; A . ~ H . E vs. A ~H.S, &4 = 3.28, P < 0 . 0 1 ) . The level of accuracy of the animals with amygdala plus hippocampus lesions decreased significantly as successive, 60, 12{} and 18{) s delays were imposed, F<~ s -- 27.58, P<:(I.001. Their performance was significantly worse in the 60 (mean {'i, c;f correct responses=70.85), 120 (mean "o ~f correct res p o n s e s = 6 2 . 8 5 ) and 180 s !mean !i, of correct responses = 56.28) delays than their own performance during the last 50 trials of training on the basic task (mean 'I~, of correct responses = 79.71: /is = 3.45, P < 0 . 0 1 ; t l s = 6 . 3 0 , P < 0 . 0 1 and tLs .= 8,63, P < 0 . 0 1 respectively). Furthermore, their performance was significantly worse at the 180 s delay than at 60 s delay (tlu=5.18, P < 0 , 0 1 ) . (See Fig. 5). The amygdala-lesioned and amygdala plus hippocampus-lesioned groups did not differ significantly in their levels of accuracy throughout the last 50 trials o f training on the basic task (mean percentage of correct responses: A.E = 78.85: A + H.E = 79.7, ..'~.~= {).33, P > 0.1 ). The performance of both groups was similar at the 60 (mean percentage of' correct responses: A . E = 76: A * H . E = 70.85,134 = 1.15, P > 0.1 ), 120 (mean percengate of correct responses: A.E = 59.7l; A + ft.E = 62.85, t~4 = 0.77, P > 0.1) and 180 s (mean percentage of correct responses: A.E = 53.71; A e H.E = 56.28, ~,~;= {).55, P > 0.1) delays.

183

M.A. Peinado-Manzano ,' Behavioural Brain Research 6 l/1994) 175- 190

2.3. Discussion

The results of this experiment suggest the ocurrence of a dissociation of hippocampus and amygdala functions in the association of a visuo-tactile configuration with reward and in the ability to remember it. The amygdala appears to be strongly involved in the learning and memory of this association. Precise analysis of the specific components of learning and memory processes involved in the associative memory task together with the examination of the actual nature of the deficits is believed to be of remarkable importance. In each trial the animals had to associate a visuo-tactile configuration with a reward and another visuo-tactile configuration with the absence of reward in the acquisition phase, retain these associations during a delay, recognize the configuration previously associated with the reward and distinguish it from the other configuration not associated with reward and finally approach the first configuration. Detailed analysis of the performance throughout the training sessions suggests that the amygdala lesions retard the formation of the association and severely impair its retention as mnesic demands are increased. All the animals, except two+ attained the first 3 levels of learning on the basic task although they required more training sessions than the control and hippocampus-lesioned animals. However+ none of the amygdala-lesioned animals reached the complete learning criterion. Furthermore, the amygdalalesioned animals exhibited a chance performance after the introduction of 120 and 180 s delays. The same pattern of performance was displayed by the most of the animals with combined damage to the amygdala and hippocampus. Because the hippocampus-lesioned animals were not impaired in the performance of the basic task, the deficits exhibited in it by the animals with amygdala plus hippocampus damage could be attributed to amygdala damage. From this analysis, I suggest that the deficit might lie in the mnemonic components involved in the elaboration and consolidation of visuo-tactile configurationreward associations. This suggestion is not in disagreement with earlier works and interpretations of the role of the amygdala. My previous works [45-48] and other studies [61], also with rats, likewise suggested that the amygdala appears to contribute to the mnemonic components involved in the acquisition of the association of sensorial stimuli with reward. Also, earlier works with monkeys support the proposed dissociation of the function of amygdala and hippocampus in the memory of objectreward associations [60]. I suggest the necessity of further experiments dissociating (1) the recognition of stimulus configurations and the distinction between them, (2) the processing of the reward and (3) the association of stimulus configurations with their reinforcing meaning. Some of

these issues have been studied in experiments described below. The hippocampus does not appear to be necessary for the association ofa visuo-tactile configuration with reward and its retention over 10 and 60 s. However, it does appear to be necessary for retention of the association over 120 s and longer periods. These results indicate a time effect associated with hippocampus damage. Similarly, it has been reported that hippocampal lesions do not affect the acquisition of a learning paradigm and that the effects of hippocampal damage are restricted to the ability to recall it over relatively long intervals [29]. Earlier works have reported the involvement of the hippocampus in other kinds of associative memory, such as visuo-olfactory crossmodal association in rats [61] and object-pair [56] and object-place [44] associations in monkeys.

3. Experiment 2 Earlier studies have not found agreement as regards the effects of hippocampus lesions on the susceptibility to interference. It has been reported that damage to the hippocampus increases this phenomenon and consequently impairs performance in different learning and memory paradigms [24,66-69]. However, other experiments have suggested that hippocampus damage does not increase the sensitivity to proactive interference [3]. In a recent work, Aggleton et al. [2] found that amygdala plus hippocampus lesions increase the sensitivity to proactive interference whereas lesions restricted to the amygdata do not produce such an increase. The present experiment evaluated the effects ofhippocampus, amygdala and amygdala plus hip-

100

•

L.Interference

80 @

,~ L

L. O

60

40

@

2.0

Z

Unop

S.op

H.E

A.E

A+H.E

GROUPS

Fig. 6. Mean percentage of correct responses in the lox~ (k) and High (H) interference conditions. (Abbreviations as in Fig. 4).

184

M.A. Peinado-Manzam) / Behavioural Brain Research 61 r1994/ 175-190

pocampus lesions on the performance of an associative memory task when proactive interference was increased. The animals were tested in two, low and high, interference conditions. In a previous unpublished work, I had observed that Wistar rats exhibit more errors when each training session on the task used in experiment 1 consisted of 9 or more consecutive trials. In view of this, I considered as a low proactive interference condition the first 8 trials of a training sesion consisting of 16 trials, and a high proactive interference condition the last 8 trials of such a training session. On the basis of earlier findings I hypothesized that damage restricted to the hippocampus and combined damage to the hippocampus plus the amygdala should increase the sensitivity to proactive interference and, in turn, should disrupt the performance of the task while damage restricted to the amygdala should not.

3. I. Materials and methods SubjecLs"

For this experiment the subjects consisted of the rats from Expt. 1.

Behavioural training The animals were tested in the T-maze used in Expt. 1. The testing procedures and the visuo-tactile stimuli were identical to those used in the basic task of the associative memory described above, with one important difference; namely, each training session consisted of 16 consecutive trials. Testing began one week after the last day of training in Expt. 1. The rats underwent a daily training session over 6 consecutive days. Statistical analysis A two-factor (Group x Interference level) ANOVA with repeated measures on the last factor was employed in the analysis of the differences between groups in terms of the number of correct responses given during the first 8 trials (low interference condition) and the last 8 trials of each training session (high interference condition). One-factor ANOVAs were used in the analysis of the differences between groups for each interference condition separately. Where necessary, the N e w m a n - K e u l s test and two-tailed t-test were used after the ANOVAs. 3.2. Results The performance of the groups throughout training under both interference conditions is summarized in Fig. 6. Before comparing the performance of the different groups, it should be taken into account that the amygdala-lesioned and amygdala plus hippocampus-lesioned groups were not at the same level of performance as the control and hip-

pocampus lesioned groups before beginning training under low- vs. high-interference conditions. The animals of all groups gave fewer correct responses during the second half of each training session. This phenomenon confirmed the occurrence of a higher interference during the last 8 trials of each session. However, this decrease in accuracy was not equal in all groups. The ANOVA revealed a group effect, G,.34 = 7.09, P < 0.001, reflecting the poor performance of the lesioned groups, P < 0.01 with the N e w m a n Keuls test; a interference level effect, F ~ 4 = 3 2 1 . 2 5 , P<0.001, and a significant interaction betwcen groups and the interference level, F'¢,,34= 19.58, P < 0.00 I, reflecting the different effects of the lesions on the performance of the task over low/high interference levels. More detailed comparisons are given below. Control groups. The unoperated control and shanloperated, H.S, A.S and A + H.S groups displayed the same performance pattern throughout the low and high interference conditions (see Fig. 6). All of them exhibited fewer correct responses during tile last 8 trials of training of each session. Statistical analyses confirmed that they did not differ significantly in their levels of performance under either interference condition (maximum t34 = 0.78, P > 0.1 for each two group comparison). Hippocampal lesions. Statistical analyses confirmed that the rats with hippocampus damage showed a level of performance similar to that exhibited by the animals of the control groups during the low interference condition (mean number of correct responses: H.E = 39.37; U n o p =: 40.5; H.S =40; H.E vs. Unop, t34 = 0,46, P > 0 . 1 ; H.E vs. H.S, t34=0.25, P>0.1). By contrast, the performance of the hippocampus-lesioned animals fell to chance level at the high interference condition (see Fig. 6). It was significantly worse than the level exhibited by the control groups (mean number of correct responses: H,E = 25,62; Unop = 36; H.S =35.4; H.E vs. Unop, t34= 5.79, P < 0 . 0 1 : H.E vs. H.S, t34 = 5.46, P < 0 . 0 1 ) .

Amygdala lesions. The amygdala-lesioned animals showed a similar performance under both interference conditions (see Fig. 6). They displayed a significantly worse performance than the comrol (mean number of correct responses: A . E = 3 I ; U n o p = 4 0 . 5 ; A.S =40.2; A.E vs. Unop, t~4=3.87, P < 0 . 0 1 ; A.H vs. A.S, .;:~4=3.75, P < 0 . 0 1 ) and hippocampus-lesioned (mean number of correct responses: H.E = 39.37, t:~4= 3.41, P < 0.01 ) groups under the low interference condition: This deficit was expected, since they were at a lower level of performance than the control and hippocampus-lesioned groups before beginning training on this task. However, the imposition of a high inteference condition had no effect on their performance. Combined lesions. The animals with amygdala plus hippocampus damage were significantly impaired with re-

M.A. Peinado-Manzano / Behavioural Brain Research 61 (I 994) 175-190

spect to the control (mean number of correct responses: A + H . E = 33.86; Unop=40.5; A + H.S = 38.6; A + H.E vs. Unop, t34=2.70, P<0.05; A + H . E vs. A + H . S , t34 = 2.06, P < 0.05) and the hippocampus-lesioned (mean number of correct responses: H.E = 39.37, t 3 4 = 2.24, P<0.05) groups under the low interference condition. Their performance was reduced to chance level during the high interference condition (see Fig. 6) and they were significantly impaired with respect to the control groups (mean number of correct responses: A + H . E = 2 4 . 3 3 ; Unop = 36; A + H.S = 34.6; A + H.E vs. Unop, t34 = 6.52, P<0.01; A + H.E vs. A + H.S, t34 = 5.74, P<0.01). 3.3. Discussion

The different nature of the deficits observed in the lesioned groups is consistent with my hypothesis and confirms an important difference between the hippocampal and amygdaloid roles in memory. Three aspects of the results suggest that hippocampal damage would increase the susceptibility to proactive interference whereas amygdala damage would not. First, the hippocampuslesioned animals performed similarly to the control groups in the low interference condition but were impaired with respect to these groups in the high interference condition. Second, the animals with lesions restricted to the amygdala showed a similar degree of accuracy throughout both the low and high interference conditions. Third, the level of performance of the animals with amygdala plus hippocampus damage under the high interference condition was lower (although not significantly worse) than the level shown by the animals with lesions restricted to the amygdala. Their greater impairment under the high interference condition might be a consequence of hippocampal damage. The present findings are consistent with earlier work reporting an increased sensitivity to interference following hippocampal damage [24,68,69]. However, at the same time, they are in disagreement with the results from other experiments [2,3]. This disagreement might be explained by differences in the nature of the task and in the proactive interference conditions. The work of Aggleton et al. [2,3] has shown that rats with hippocampus lesions have a similar performance to that shown by control animals, whereas rats with amygdala plus hippocampus lesions do not, when the stimuli are repeated in the same training session in a recognition memory task. By contrast, I have evaluated the effect of increasing the amount of information presented during the same training session in an associative memory task. It has also been reported that lesions restricted to the amygdala are not associated with an increased sensitivity to the interference [2]. However, the lack of an increased

185

sensitivity to interference shown by animals with amygdala damage in the present experiment should receive further attention because they were already impaired in the performance of the task under the low interference condition.

4. Experiment 3 In a previous experiment [47], I found that amygdala lesions disrupted the acquisition of a multiple-trial visualstimulus/magnitude-of-reinforcement discrimination. It has been also reported that amygdala lesions impair the response to the change in the magnitude of reinforcement in rats [18,19,27] and in monkeys [57] and the formation of the association between a place and different magnitudes of reinforcement [28,30]. Taken together, the findings from these different experiments are suggestive of a general role of the amygdala in processing and remembering the association of stimuli with different amounts of reward. Two major questions arise concerning this role. The general involvement of the amygdala in the association of stimuli with reward in the different learning and memory systems should be further studied. The selective contribution of the amygdala vs. the hippocampus to this function is also unknown. In the present experiment I evaluated the effects of amygdala, hippocampus or amygdala plus hippocampus lesions on the performance of a one-trial task which requires the association of visuo-tactile configurations with different magnitudes of reinforcement. On the basis of the results of above-mentioned works, I hypothesized that amygdala and amygdala plus hippocampus-lesioned animals should exhibit an impaired performance of the task. A lack of impairment was predicted for rats with hippocampus damage. 4.1. Materials and methods Subjects" For this experiment the subjects consisted of the rats from Expt. 2. Behavioural training Testing began a week after completion of Expt. 2. The animals were trained in the T-maze as in the previous experiments. The testing procedures and visuo-tactile configurations were identical to those used in the associative memory basic task, described above, with one important difference: the magnitude of reinforcement was changed over three experimental conditions in order to decrease the saliency of the discrimination. A daily training session consisting of 5 trials with a 60-s intertrial interval was given, 5 days a week, to each animal.

186

M.A. Peinado-Manzam~ ~Behavioural Brain Research 61 ~1994) 175--t 90

Condition I. This initial stage closely matched the training procedures used in the basic task. The animals received 1 standard 45-rag pellet of food for the rewarded visuo-tactile configuration and no reward was associated with the other configuration during the acquisition phase. In the test phase, the animals were rewarded with one standard 405-mg pellet if they chose the stimulus configuration rewarded in the acquisition phase. The rats underwent a total of 20 training sessions. Condition H. Twenty-four h after completion of condition I the animals were tested for their ability to associate a large reward with a visuo-tactile configuration and a small reward with another visuo-tactile configuration and to remember which configuration had been associated with the large reward. In the acquisition phase, the rats were allowed to eat one/five 45-mg pellets for the configuration associated with small/large reward respectively. In the test phase, the animals received one standard 405-mg pellet for having chosen the stimulus configuration associated with the large reward in the acquisition phase, but were unrewarded if they chose the configuration associated with the small reward. The animals received a total of 40 training sessions. Condition III. The task was made more difficult during this stage since the small reward was increased from one to two 45-mg pellets, during the acquisition phase, whereas the large one (five 45-mg pellets) was not changed. Once again, during the test phase the animals received one standard 405-mg pellet if they chose the stimulus configuration associated with large reward in the acquisition phase but were unrewarded for having chosen the configuration associated with the small reward. Training began 24 h after the last training session of Condition II. The animals underwent another 40 training sessions.

Statistical analysis

A two-factor (Group x Magmtude of reinlbrcement) ANOVA with repeated measures on the last factor was employed in the analysis of the differences between groups in terms of the percentage of correct responses given during the last 10 training sessions under each experimental condition. A three-factor (Group x Magnitude of reinforcement x Block of training sessions) ANOVA with repeated measures on the last two [',actors was employed in the analysis of the differences between groups in terms of the percentage of correct responses given during the four blocks of 10 training sessions of experimental conditions I1 and Ill. The Newman-Keuls test and two-tailed t-test were used whenever necessary after the ANO\,'As. 4.2. Resuhs

The pertbrmance of the groups throughout the three experimental conditions is summarized in Table 2 and Fig. 7. As expected, all groups maintained throughout experimental condition I a level of accuracy similar to that reached during training in the basic task in Expt. I. Since the saliency of the discrimination was decreased in the acquisition phase of each trial, the level of performance dropped near to chance level for all groups under experimental conditions II and II1. However, it selectively improved for each group throughout training under these conditions. The Group x Magnitude of reinforcement ANOVA revealed a group effect, Fe,,34 = 11.57, P<:0.001

100 q

Table 2 Mean (_+ S.E.M.) percentage of correct responses given throughout training on magnitude of reinforcement condition II (C.II) and III (C.III) over successive blocks (B1, B2, B3, and B4) of 10 training sessions

! L.

B1

B2

B3

Condition I Condition tl Condition Iit

80

O

Group

[] [] ~

60

B4 O

Unop: S.op: H.E: A.E: A+H.E

C.II C.II| C.II C.III C.II C.III C.II C.III C.II C.I|I

59.50+4.11 61.50_+2.21 56.66_+2.65 56.93_+2.13 55.50_+3.92 56.50_+4.10 52.28_+2.32 47.71_+1.59 53.14_+3.26 52.85 _+2.98

74.50_+2.75 79.00_+1.9t 78.13_+1.91 72.53_+2.14 74.00_+1.96 83.00_+ 1.69 52.57_+3.10 52.28_+2.02 55.42_+3.79 57.42_+4.59

82.00_+3.46 81.50_+1.50 79.20_+1.16 84.66_+1.19 77.75_+2.21 78.50_+ 1.50 56.00_+3.90 49.14_+1.68 61.42_+5.42 58.28 _+4.62

83.00_+1.91 79.00_+2.64 80.80_+1.08 82.80_+1.11 86.00_+1.36 84.25_+ 1.93 64.85_+1.50 5Z00_+2.43 63.71_+4.88 58.85_+6.39

t_

8

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O

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20

0

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~ 7 ~

-¸ =

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GROUPS

Unop, unoperated control group; S.op, sham-operated groups: HE, hippocampus lesioned group; A.E, amygdala-lesioned group; A + E.E, amygdala plus hippocampus-lesioned group.

Fig. 7. Mean percentage of correct responses in the last 10 training sessions for the magnitude of reinlbrcement condition L II -and I11. (Abbreviations as in Fig. 4).

M.A. Peinado-Manzano / Behavioural Bra#t Research 6I (1994) 175-190

which reflected the poor performance of the amygdalalesioned and amygdala plus hippocampus-lesioned groups throughout the three experimental conditions, P<0.01 with the N e w m a n - K e u l s test, a magnitude of reinforcement effect, F2.~,~ = 189.25, P<0.001, and a significant interaction between groups and magnitude of reinforcement, F~z.~,~=61.8, P<0.001, confirming that the impairment exhibited by the amygdala-lesioned and amygdala plus hippocampus-lesioned groups, with respect to the control and hippocampus-lesioned groups, had changed as a function of the different magnitudes of reinforcement. The Group x Magnitude of reinforcement x Block of training sessions ANOVA revealed a group effect, f ( ~ . 3 4 = 16.86, P<0.001, which reflected the poor performance of the amygdala-lesioned and amygdala plus hippocampus-lesioned groups throughout experimental conditions II and IIL P<0.01 with the N e w m a n - K e u l s test; a block of training session effect, F4,102 = 159.33, P < 0.001, reflecting the improvement shown by the groups throughout the training sessions; a significant interaction between groups and blocks of training sessions, F~, 1 < - 7.78, P<0.001, which confirmed the occurrence of different degrees of improvement throughout the training for the different groups; a non-significant magnitude of reinforcement effect ( F < 0 ) ; a non-significant interaction between groups and magnitude of reinforcement, F~, 34 ~ 2.2, P>0.05; a non-significant interaction between magnitude of reinforcement and blocks of training sessions ( F < I), and a non-significant group x magnitude of reinforcement× block of training sessions interaction, Fis.L<=1.27, P>0.1. More detailed comparisons are given below. C)mtrol groups. The unoperated control and shamoperated, H.S, A.S and A + H.S, groups, showed the same perfk~rmancc pattern under all three experimental conditions on the magnitude of reinforcement (see Table 2 and Fig. 7). Statistical analyses confirmed that they did not differ significantly in their levels of accuracy (maximum t~a =/I.97, P > 0.1 for each two group comparison). All of them learned both new magnitude of reinforcement discriminations and recovered a level of accuracy similar to that shown under experimental condition I. Statistical analyses revealed that the lesioned groups showed a different performance during training under the different magnitude of reinforcement conditions. Hippocampal lesions. Animals with hippocampus damage were unimpaired in both new magnitude of reinforcement discriminations with respect to the control groups (Condition II, mean percentage of correct responses: H.E=86: Unop-83: H . S = 8 2 . 4 ; H.E vs. Unop, t~4 = 0.80, P > 0 . 1 ; H.E vs. H.S, t m = 0.93, P > 0.93; Condition III, mean percentage of correct responses: H.E=84.25: U n o p = 7 0 : H . S = 8 2 . 4 ; H.E vs. Unop,

187

t34 = 1.15, P > 0 . 1 ; H.E vs. H.S, t34= 0.45, P>0.1). (See Table 2 and Fig. 7). Amygdala lesions. This damage severely disrupted the acquisition of both new magnitude of reinforcement discriminations with respect to the control (Condition II, mean percentage of correct responses: A.E=64.85; Unop = 83; A.S = 80.4; A.E vs. Unop,/34 = 4.02, P < 0 . 0 1 ; A.E vs. A.S, t 3 4 = 3.37, P<0.01; Condition III, mean percentage of correct responses: A . E = 52; U n o p = 7 9 ; A.S =83.2; A.E vs. Unop, t34=4.95, P<0.01; A.E vs. A.S, t34 = 5.87, P < 0.01) and hippocampus-lesioned (Condition II, mean percentage of correct responses: H.E = 86, t 3 4 = 4 . 8 3 , P < 0.01; Condition III, mean percentage of correct responses: H.E = 84.25, /34 = 6.13, P < 0 . 0 1 ) groups. The amygdala-lesioned rats could not discriminate 1 piece from 5 pieces of food, nor 2 pieces from 5. The level of accuracy reached during condition I fell nearly to chance level as the saliency of the discrimination was reduced in conditions II and III and had still not been recovered at the end of training (see Table 2 and Fig. 7). Combinedlesions. Amygdala plus hippocampus-lesioned animals were severely impaired in the acquisition of both new magnitude of reinforcement discriminations. Statistical analyses showed that they exhibited a worse performance than the control (Condition If, mean percentage of correct responses; A + H.E = 63.71; Unop = 83; A + H . S = 7 9 . 6 ; A + H . E vs. Unop, t~4=4.14, P<0.01; A + H.E vs. A + H.S, t34 = 3.31, P<0.01; Condition llI, mean percentage of correct responses: A + H.E = 58.85; U n o p = 79; A + H.S = 82.8; A + H.E vs. Unop, t34= 3.64, P<0.01; A + H . E vs. A + H . S , t~4=4.46, P < 0 . 0 1 ) and hippocampus-lesioned (Condition II, mean percentage of correct responses: H.E = 86, t~4-4.95, P<0.01; Condition IlI, mean percentage of correct responses: H.E=84.25, t34=4.82, P < 0 . 0 1 ) g r o u p s . Their level of accuracy fell to near chance level as the saliency of the discrimination was reduced and had still not been recovered at the end of training (see Table 2 and Fig. 7). They were unable to discriminate 1 piece from 5 pieces & f o o d , nor 2 pieces from 5.

4.3. Discussion The failure of rats with amygdala or amygdala plus hippocampus damage to learn the task when the difference between the amount of reward associated with each stimulus configuration was reduced confirms the contribution of the amygdala to the association of visuo-tactile configurations with different magnitudes of reinforcement. This conclusion is not in disagreement with the earlier work [28,30,47]. The unimpaired performance of the same task that followed hippocampus damage, in the present experi-

188

M.A. Pe#zado-Manzano

Behavkmral Brab2 Research 6l ~1994) 175-190

ment, supports my hypothesis and indicates that the hippocampus is not involved in such processes. The observed impairment could stem from a number of different deficits. Possible critical deficits include a failure to identify and distinguish different visuo-tactile configurations; an impaired ability to identify and distinguish different amount of reward, and the loss of the ability to associate visuo-tactile configurations with different magnitudes of reinforcement and/or remember such an association. Detailed analysis of the performance of the animals with amygdala or amygdala plus hippocampus damage throughout this experiment suggests that the critical deficit may lie in the formation of the association of visuo-tactile configurations with reward and in the ability to remember this association, particularly when differing values of reinforcement must be distinguished.

5. General discussion The experiments carried out in this work form part of a series of studies that have attempted to evaluate the proposal that the amygdala and the hippocampus can be functionally dissociated with respect to learning and memory processes. The results of previous experiments [48] suggest that the involvement of the amygdala and the hippocampus in episodic memory appears to be dissociated in terms of the spatial vs. non-spatial nature of the information to be processed. The data from the present experiments also support the occurrence of a dissociated and non-conjoint intervention of both structures in memory of the association of sensorial stimuli with reward. Both appears to be involved in this kind of associative memory, but in different stages and components of the process. Taken together, the effects of the amygdala, hippocampus and amygdala plus hippocampus lesions appear to indicate: (1) a critical role of the amygdala in learning and memory of the sensorial stimuli-reward association and in processing magnitude of reinforcement; (2) a selective role of the hippocampus in recalling the information over relatively long periods of time and (3) an increased susceptibility to interference after hippocampus damage. The amygdala appears to be particularly involved in the elaboration and consolidation of the association of visuotactile configurations with reward. A further question arises as to whether it is necessary for all kinds of associative memory or whether it is only involved in the recall of certain particular associations. Similarly, previous works [45-49] indicate a selective contribution of the amygdala to the mnemonic components required for processing the affective meaning of the sensorial information, but not to the association of the spatial information with reinforcement. Earlier reports also support this proposal

ibr monkeys [38,44,51,60]. However, results tYom other earlier studies do not fit in well with all the components of this hypothesis. Firstly, in rats the amygdala does not appear to be necessary for the performance of tasks which require processing of the motivational meaning of visuotactile [2,52], olfactory [9] and visuo-olfactive information [61 ]. Secondly, it has been reported that the amygdala appears to be involved in the formation of place-reward associations [ 28,30]. The above-mentioned discrepancies could be explained by two alternative or even complementary proposals. The critical role of the amygdala in processing the affective meaning of any kind of information when it raises high levels of arousal would be one possible suggestion. Its particular contribution to processing different magnitudes of reinforcement would be another complementary hypothesis. In the present work, animals with amygdala lesions severely decreased their level of accuracy to chance pertbrmance when they were tested on the same associative memory task but one requiring the discrimination of different magnitudes of reward. Previously, I had l\~und that amygdala damage impairs the association of visual stimuli with different magnitudes of reinforcement in a different learning paradigm [47]. Additionally, it has been reported that amygdala damage disrupts the response to changes in the magnitude of reinforcement in rats [18,1%27] and in monkeys [ 57 ]. Furthermore, works that have reported an involvement of the amygdala in the formation of placereward associations [28,30] used tasks in which the animals had to distinguish between several magnitudes ot reward. In view of the foregoing data, I propose the possible involvement of the amygdala in, firstly, the association of sensorial stimuli with reinforcement and, secondl?. in processing the magnitude ot reinforcement associated with any kind of information. Further research concerning the dissociated or complementary role of the amygdale in both kinds of processes is buggested. The hippocampus appears to be selectively involved ir recalling sensorial information o~er longer periods of time An important question concerning the role of the hippocampus in memory arose from this and previous work [48] that is, the possible relationship between: (1) its selectiw involvement in learning and memory of spatial vs. non spatial inlk)rmation and (2) its dissociated rote in recallint information after short vs. long intervals of' time. Its se lective contribution to processing and storing spatial in formation but not non-spatial information is supported b' other experimental data [3,26,36,37,48,53,59]. Earlier re search also indicates selective deficits in recalling infor mation over long periods of time alter hippocampus dan1 age in rats [29,65,69] and monkeys [14,43]. Is th hippocampus involved in recalling any kind of informatio over long periods of time? This question arises from thi

M.A. Pe#Tado-Manzano / Behavioural Bra#7 Research 61 (1994) 175-190

s t u d y as a m a j o r problem to b e a d d r e s s e d in f u r t h e r research. A s e c o n d m a j o r f i n d i n g in this s t u d y c o n c e r n i n g t h e m n e s i c role o f t h e h i p p o c a m p u s w a s t h e p o o r p e r f o r m a n c e of the hippocampus-lesioned

rats under the high interfer-

e n c e c o n d i t i o n . T h e lack o f c o n s i s t e n c y w i t h p r e v i o u s w o r k [3] s u g g e s t s t h e n e c e s s i t y o f f u r t h e r r e s e a r c h i n t o t h e p o s sible c a u s e s o f t h e p r o a c t i v e i n t e r f e r e n c e p h e n o m e n o n a n d the possible different roles of the hippocampus

in its re-

lease.

6. Acknowledgements This work was supported by the Direcci6n General de lnvestigaci6n Cientifica y T6cnica, Grant PS88-0038.

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