Effects of intra-amygdala injections of NMDA receptor antagonists on acquisition and retention of inhibitory avoidance

Brain Research, 585 (1992) 35-48 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

35

BRES 17793

Effects of intra-amygdala injections of NMDA receptor antagonists on acquisition and retention of inhibitory avoidance M u n s o o Kim and J a m e s L. M c G a u g h Centerfor the Neurobiology of Learning and Memory and Department of Psychobiology, Universityof California, Irvine, CA 92717 (USA) (Accepted 21 January 1992)

Key words: Amygdala; Inhibitory avoidance; Long-term potentiation; NMDA receptor; AP5; CPP; MK-801

These experiments examined the effects of intra-amygdala injections of NMDA receptor antagonists on the acquisition and retention of inhibitory avoidance. In Expt. l, rats received bilateral intra-amygdala injections of the NMDA antagonists D,L-AP5 (1-10 ~.g), D-AP5 (0.03-1 /Lg), CPP (0.125 or 0.375 p,g), or MK-801 (0.2 or 0.5/Lg) prior to training in a continuous multiple-trial inhibitory avoidance (CMIA) task. Acquisition of the task was not significantly affected by any of the drug injections. In contrast, all three competitive antagonists, D,L-APS, D-AP5 and CPP, produced dose-dependent impairment of 48 h retention performance. Although the MK-801 injections did not significantly impair retention performance, the retention scores of the 0.5 /zg MK.801 group were bimodally distributed, indicating retention impairment in a subgroup of the animals given that dose. Intra-amygdala injections of 3 or 10 ~g D,L-AP5 did not affect footshock sensitivity (Expt. ll) or locomotor activity (Expt. lID and their retention-impairing effects were not due to induction of state dependency (Expt. IV). The retention-impairing effects of intra-amygdala injections of NMDA antagonists were not due to diffusion of the drugs dorsally: injections of I ~.g D-AP5 into the striatal area directly above the amygdala impaired acquisition but not retention performance (Expt. V). The retention-impairing effects of 1 I~g D-AP5 or 0.5/~g MK-801 were attenuated by giving additional training to the animals shortly after receiving intra-amygdala injections (Expt. VI), The implications of these findings for hypotheses concerning amygdala function in learning and memory are discussed.

INTRODUCTION Extensive evidence indicates that the amygdala plays an important role in learning and memory 21'32'4"~'7°. Lesions of the amygdala impair the acquisition and retention of a variety of learning tasks7°. Pharmacological manipulation of neurotransmitter systems in the amygdala, including the noradrenergic 29's°, GABAergic 7, or opioid peptidergic 2s'4L56 systems, affects inhibitory avoidance (IA) learning and Pavlovian fear conditioning. Recent findings suggest that the N-methyl-D-aspartate (NMDA) receptor system in the amygdala is also involved in aversively-motivated learning: microinfusion of an NMDA receptor antagonist, D,L2-amino-5-phosphonovalerate (D,L-APS), into the basolateral amygdala impairs one-trial inhibitory avoidance learning 49, Pavlovian conditioning and extinction of fear-potentiated startle 9'24'57.

The present experiments investigated further the involvement of amygdala NMDA receptors in the acquisition and retention of inhibitory avoidance. All the studies, to date, examining the role of NMDA receptors in IA learning have used one-trial IA tasks I'j,22''~°,4'J,~s'7'~.Thus it is not known whether the effects of NMDA antagonists administered prior to training are due to disruption of acquisition or to impairment of memory storage processes. To address this issue, rats in these experiments were given intraamygdala injections of selective NMDA antagonists prior to training to criterion on a multiple-trial inhibitory avoidance task and were tested for retention 48 h later. Control experiments examined the effects of the NMDA antagonists on footshock sensitivity and locomotor activity, drug-induced state-dependency, and the anatomical specificity of the intra-amygdala drug treatment.

Correspondence: M. Kim, Department of Psychiatry, Yale University School of Medicine, Abraham Ribicoff Research Facilities, Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508, USA.

36 MATERIALS AND METHODS

Subjects A total of 335 male Sprague-Dawley rats (approximately 60-days old, 200-250 g on ~rrival, Charles River Labs.) were used. They were individually housed and maintained on a 12-h light/dark cycle (lights on at 07.00 h) with food and water available ad libitum and were acclimatized to laboratory conditions for at least 5 days before the surgery. The behavioral studies were begun 5 or 6 days after the surgery. Each rat was handled once for 4 rain, 1-2 days prior to the behavioral training. All experiments were conducted between 13.00 and 19.00 h.

Surgery Amygdala cannulae implantation. The rats were anesthetized with nembutal (50 mg/kg, i.p.) and given atropine sulfate (0.4 mg/kg, i.p.). Supplemental doses of chloral hydrate (20-60 me) were given as needed to maintain anesthesia. Stainless-steel guide cannulae (23 gauge, 15 ram) were placed bilaterally in the dorsal surface of the amygdala. The stereotaxic coordinates relative to bregma were: A.P. -2.3 ram; M.L. + 4.4 ram; D.V. -5.2 mm from dura; with the nose bar at -3.3 mm from the interaural line~'. Surgical screws placed in the skull above the frontal and posterior cortices served as anchors. The cannulae were affixed to the skull with dental cement. Stylets (i.5 ram) made of 00 insect dissection pins were inserted into the guide cannulae and remained there at all times except during injections. Immediately after surgery the animals received an intramuscular injection of penicillin (0.1 ml) and were maintained in a temperature controlled chamber (32-35°C) until recovery from anesthesia. Striatum cannulae implantation. Cannulae were implanted bilaterally into the striatum using the same general procedures described above. The striatum coordinates were: relative to bregma, A.P. -2.3 mm; M.L. ± 4.4 mm: D.V. - 3.0 mm from dura: with the nose bar at - 3.3 mm from the interaural line~,

Histology The animals were sacrificed with an overdose of nembutal or chloral hydrate, and their brains removed and stored in I0% formalin for at least 5 daD, The brains were then sectioned (40 ,am), and the slices containing the cannula tracts were stained with Cres~t violet. Only animals with both of the injection needle tips located in the amygdala were included in the statistical analysis. Figures for histological verification are shown only for Expts, I and V, The histological results from other experiments were highly comparable to those of Expt. !.

Injection procedures The animals received bilateral injections into the amygdala or striatum 3 or 10 min before the behavioral testing. The rat was gently restrained by hand, the stylets were withdrawn from the guide cannulae, and 30-gauge injection needles were inserted to a depth of 2 mm beyond the tips of the guide eannulae. The injection needles were connected via PE-20 polyethyelene tubing to 10-~tl Hamilton microsyringes driven by an automated syringe pump. The injections were delivered concurrently in a volume of 0,5 ~tl per syringe over a period of 37 s. After the injections the needles were retained in the guide cannulae for an additional 40 s to increase diffusion of the solution into the tissue, The stylets were then replaced in the guide cannulae and the rat was returned to its home cage.

Behat,ioral testing Continuous multiple.trial inhibitory avoidance (CMIA) task. The rats were trained on a trough-shaped alley (91 cm long, 15 cm high, 20 cm wide at the top and 6.4 cm wide at the bottom)51 consisting of two compartments separated by a sliding door. The starting compartment (31 cm long) was illuminated by a tensor lamp which provided the only illumination in the room. The stainless-steel floor of the darkshock compartment (60 cm long) was divided into two sections by a 0.5 cm slit in the the middle of the floor. The apparatus was located in a sound-attenuated room. A continuous multiple-trial inhibitory avoidance training procedure was used. On the first acquisition trial, the rat was placed in the starting compartment, facing the closed door. When the rat turned around, a timer was started, the door was opened and left open during the entire training period. When the rat stepped into the shock compartment (with all four paws), a low-intensity footshock (0.55 mA, 60 Hz) was delivered until it escaped back to the starting compartment. The second acquisition trial began, without interruption (i.e. training was continuous), immediately after the escape response, if the rat remained in the starting compartment for 100 s (the acquisition criterion), the training was terminated and the score of 1 was recorded as the number of trials to criterion. If the rat entered the shock compartment again within 100 s, footshock was delivered until the rat escaped back into the starting compartment. Training was continued until the rat remained in the starting compartment for 100 consecutive s. The total number of trials (i.e. the number of entries into the shock compartment) required for the rat to reach the acquisition criterion was recorded. For most rats, the training was completed within 5 rain. Upon reaching the acquisition criterion, the rat was removed from the apparatus and returned to its home cage. Prior to the training of each rat, the alley of the apparatus was cleaned with a 20% EtOH solution. On the retention test 48 h later, the animals were placed in the starting compartment, as in the initial training, and the latencies to enter the shock compartment (maximum of 600 s) were recorded. Footshock was not administered on the retention test except as indicated below. This task provided three measures: the step-through latency on the first trainin~ trial (training latency), the number of trials to criterion (NTC), and the step-through latency on the retention test (retention latency), The first two served ;is mea,~ures of acquisition performance, and the third served as a measure of retention performance, ,4dditiomd traintt~g procedurt,, In Expt, VI, the rats were given additional training, Rats in this experiment were first trained and tested according to the CMIA procedure described above, Immediately after the 48 h retention test trial, the rats were given additional training and, after another 48 h, they received a second retentio~ te~t (see Table I). On the first retention test, immediately after the rat entered the shock compartment, a footshock (0.55 mA) was delivered until the rat escaped to the starting compartment. The step-through latency measured on this trial is designated as the first retention latency. Thus, the additional training started, without interruption, immediately after the first retention test, and was given in two consecutive phases; re-learning and escape training, in the re-learning phase, the rat was retrained to the criterion of remaining in the starting compartment for 100 consecutive s and the number of trials required to reach the criterion was recorded. If the rat remained in the safe compartment for 600 s on the first retention test, the rat was removed from the IA apparatus at the end of the test, placed in the shock compartment and given a footshock until it escaped to the safe compartment. The rat was ti;en left in the safe

Dtlags The drugs used in this study were the NMDA antagonists D,L-AP5 (Research Biochemical Inc,), D-AP5 (Tocris Neuramin), 34( + / - )2-carboxypiperazin.4.yl)propyl-|.phosphonic acid (CPP, Tocris Neuramin), and (+)-5-methyl-10,11-dihydro.SH.dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, Research Biochemical Inc.). The drugs were dissolved in phosphate buffer (0,105 g of PO,,NaH2.H20 and 0,029 g of PO4Na2H in 100 mi of saline solution, pH 7.0-7.4) immediately before experiments. The drug dosages indicated are the amounts injected into each amygdala.

TABLE I

Sequence of treatment, initial- and additional training, and retention test Day 1 Day 3 Day 5

intra-amygdala drug injection ~ CMIA training intra-amygdala drug injection --* first retention test --* additional training second retention test

37 compartment for 100 s in order to ascertain the acquisition criterion was reached and a score of 0 was given as the number of trials required for re-learning. This procedure was used to prevent extinction of the IA response that may have occurred as a consequence of the animal's remaining in the IA box. The escape training phase started immediately after the re-learning phase. After reaching the 100 s acquisition criterion on re-learning, the rat was removed from the starting compartment and placed directly in the middle of the shock compartment facing away from the starting compartment with the door closed. The door was then opened and a footshock (0.55 mA) was delivered until the rat escaped to the safe compartment. If the rat entered the shock compartment within 60 s, footshock was delivered again until it escaped to the safe compartment. It was retained in the safe compartment during the 60 s inter-trial interval. Each rat received five escape training trials. The time taken from the beginning of the first retention test to the end of escape training ranged from 8 to 18 min. The second retention test was given 48 h later using the same procedures used for the fi~t retention test, and the step-through latency (the second retention latency) was recorded. As is shown in Table !, drug injections were administered before training on day 1 and before testing/additional training on day 3, hut not before testing on day 5. Footshock sensitit,ity test This test used the same IA apparatus as that used in Expt. I in order to make the footshock stimulation conditions as similar as possible to those used in the CMIA training. For this test, the door was closed during the entire testing period and the shock compartment was illuminated with a tensor lamp to allow observation of the animal's reaction to footshock. 3 min after intra-amygdala drug injections, the rat was placed in the shock compartment facing away from the door. Footshock (0.8 s) of various intensities was given in

the following pseudo-random order: 0.15, 0.25, 0.1, 0.3, 0.2, 0.35 mA (repeated once more in this order). The interval between footshocks varied from 10 to 30 s. If an animal showed flinch response to a given intensity of footshock, it was given a score of 1, and if no response, a score of 0 was given. Since the series of footshock was given twice, the possible scores were 0, I or 2 for each footshock level. Locomotor actiuity test The locomotor activity test was conducted in a trough-shaped Y-maze s located in a dimly illuminated room. Each alley was 45 cm long, 18.5 cm high, 4 cm wide at floor level and 19 cm wide at the top. Each alley was divided into two sections by a 0.5 cm slit in the middle of the stainless steel alley floor. Sliding doors were located between the two sections of each alley. The maze was covered with a Y-shaped clear Plexiglas top and the end of each alley was illuminated by a light located behind translucent white Plexiglas. The tests were conducted on three consecutive days. On day I, the rats were habituated to the Y-maze. They were put in the starting alley of the maze, facing a sliding door with the door closed. After 5 s, the door was opened and the rat was allowed to explore the maze for 5 rain. If the rat did not leave the starting section within 2 min, it was gently guided into the adjacent section. This was required for fewer than 10% of the animals. On day 2, the locomotor activity was measured for 4 min by recording the number of sections that the rat entered. An entrance was recorded when the rat put all four paws on one section. On day 3, 3 min after intra-amygdala drug injections, the rats were placed in the starting section and allowed to explore the maze and the number of sections of the maze entered on three consecutive 2 rain sessions (i.e. a total of 6 min) was recorded. The successive choices of the arms that the rat entered and the spontaneous alternation in entering the three arms were also recorded. A spontaneous alternation was recorded in the following way: if the rat entered arm C after entering arms A and B, the

A

-1.80

-1,80

-2.12

-2.12 -2.30

-2.56 -2.30

-3.14

-2.56

-3.30

Fig. 1. Location of the tip of the injection needles in the amygdala (A) in Expt. I, or in the striatal area (B) in Expt. V.

38 entrance of arm C was counted as an alternation: re-entrance of arm A or B was counted as a non-alternation. A choice was defined as entering an arm with all four paws after reaching the center of the Y-maze from any arm. The percent spontaneous alternation was calculated by dividing the number of alternations by the total number of choices.

Statistical anab'sis Data obtained in the CMIA task are expressed as mean + S.E.M. for the training latencies and NTC, and as median and interquartile ranges for the retention latencies. ANOVA and two-tailed t-tests were performed on the training latency and NTC data. The Kruskal-Wallis one-way ANOVA, the two-tailed Mann-Whitney U-test, and the Wilcoxon test for two matched groups "w were used for analyses of the retention latency data since the range of the retention latencies was restricted by the maximum score of 600 s. Also, non-parametric Friedman and X 2 tests were performed on the footshock sensitivity test data since the reaction score of a rat at each shock level fell into one of three categories (0, 1 or 2). A two-way repeated measures ANOVA was performed on the locomotor activity test data. P values of less than 0.05 were considered significant.

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i~

20

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t

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Fig. 2. Effects of pre-training intra-amygdala injections of D,L-AP5 (A), D-AP5 (B), CPP (C), or MK-801 (D) on the training latency in Expt. !. * Different from the 0.3/zg D-AP5 group, P < 0.02.

RESULTS

Experiment i This experiment examined the effects of intraamygdala injections of three competitive NMDA antagonists, D,L-AP5, D-AP5 an~ CPP, and a non-competitive NMDA antagonist, MK-801, on acquisition and retention of the CMIA response. D,L-AP5 and D-AP5 injections were administered 3 min before the CMIA training. Since some non-specific drug effects were found when 3-min injection-testing interval was used in Expt. IV below (also see Discussion), CPP and MK.801 were injected 10 rain before training. Doses affecting memory without inducing apparent behavioral side effects (except the dose of 10/zg t~,L-APS) were selected on the basis of pilot studies. The doses used were: 0 (n - 20), 1 (n = 9), 3 (n = 18) and 10/~g (n = 14) of n,L-AP5; 0 (n - 14), 0.03 (n = 11), 0,1 (n = 10), 0.3 (n = 13) and 1/zg (n = 16) of D-AP5; 0 (n = 7), 0.125 (n = 7) and 0.375/zg (n = 5) of CPP; 0 (n = 10), 0.2 (n - 8 ) and 0.5 ~g (n - 12) of MK-801. Control animals (0 ~g groups above) were injected with the vehicle (phosphate buffer) only. No injections were given prior to the retention test. The D,L-AP5 and MK-801 experiments were performed twice and the D-AP5 experiment was performed three times. The data were pooled (n's indicated above are the pooled n's) for statistical analysis of each experiment. Figure 1A shows the location of injection needle tips in the animals given intra-amygdala injections of vehicle or D,L-AP5. In most animals, the tips were located in or near the lateral part of the central nucleus adjacent to the lateral and basolateral nuclei. Considering the possible extent of diffusion of the drug and the volume of injection (0.5/zi/each side) used in this study, it is likely that the drug affected the central:

lateral and basolateral nuclei. The histological results (not shown) from the experiments using D-AP5, CPP and MK-801 were highly comparable to those shown in Fig. 1A. On the first trial of the CMIA training, all rats entered the shock compartment within 60 s. The rats typically escaped from the footshock by re-entering the starting compartment withi. 2 s after the footshock onset, It should be noted that some of the rats in the I0/~g D,L-AP5 group displayed increased sniffing and rearing, and sometimes lost balance and fell during the CMIA training. As is shown in Fig. 2, the training latencies were not affected by the injections of D,L-AP5 (~,,~7 < I), CPP (F2,t4 = 1.088, P > 0,3), or MK-801 (F2,27 < I), but were significantly affected by D-AP5 (F4,s~ = 2.567, P < 0.05). The training latencies of the 0.I/~g D-AP5 group differed significantly from those of the 0.3/zg D-AP5 group ( t 2 t - 2,677, P<0,02), This effect of D-AP5 seems to be of questionable significance since no consistent dose-reponse relationship was found and other NMDA antagonists did not produce comparable effects, As is shown in Fig, 3, the NTC (number of trials to criterion) was not affected by injections of D,L-AP5 (F3,.s7 = 2.295, 0.05 < P < 0.1), D-AP5 (F4,59 < 1), CPP (F2,14 < 1), or MK-801 (F2,27=2.586, 0.05 < P < 0 . 1 ) . Thus, the training latency and NTC data indicate that intra-amygdala injections of NMDA antagonists prior to training did not significantly affect acquisition of the CMIA response. As is shown in Fig. 4, pre-training intra-amygdala injections of the competitive NMDA antagonists produced dose-dependent impairment of retention of the

39 lO

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Fig. 3. Effects of pre-training intra-amygdala injections of D,L-AP5 (A), D-AP5 (B), CPP (C), or MK-801 (D) on the number of trials to criterion in the CMIA training in Expt. I. There is no significant treatment effect.

CMIA response. Kruskal-Wallis one-way ANOVAs revealed that the retention latencies were significantly affected by injections of D,L-AP5 (H3 = 24.689, P < 0.001), D-AP5 (//4 = 18.1, P < 0.002), and CPP ( H 2 = 12.819, P < 0.002). Subsequent Mann-Whitney U-tests revealed significant differences between the retention latencies of the following groups: in the D,L-AP5 experiment, vehicle vs. 3 / z g (U = 74, P < 0.002), vehicle vs. 10/zg (U = 17,5, P < 0.001), 1 /zg vs. i 0 / ~ g (U = 18, P < 0.005), and 3 ~g vs. 10 ~ g (U - 60.5, P < 0.02); in the D - A P 5 experiment, vehicle vs. 1 /zg ( U = 27.5, P < 0.001), 0.03 p,g vs. 1 /zg (U - 32.5, P < 0.01), 0.1 ~ g vs. 1 ~ g ( U - 2 5 . 5 , P < 0.01), and 0,3 ~g vs. 1 ~ g

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Fig. 4. Effects of pre-training intra-amygdala injections of D,L-AP5 (A), D-AP5 (B), CPP (C), or MK-801 (D) on the retention latency in Expt. I. Each dot in D represents the retention latency score of each animal, a, different from the vehicle group; b, different from the 1 and 3/zg D,L-AP5groups; c, different from the other four gro!ms in the D-AP5experiment. See text for P values.

(U = 37, P < 0.01); in the CPP experiment, vehicle vs. 0.125 /zg (U = 2, P < 0.005) and vehicle vs. 0.375 /~g (U = 0, P < 0.005). In contrast with the effects of the competitive NMDA antagonists, MK-801 injections did not significantly affect the retention latencies ( H 2 = 2.516, P > 0.25). However, as shown in Fig. 4D, the retention latency scores of the 0.5 p,g MK-801 group were bimodally distributed. Of 12 rats in this group, 5 rats scored the maximum latency of 600 while the other 7 rats scored under 100 (range 22-87). This bimodal retention performance was seen in both experiments using MK-801. The retention scores of the vehicle, 0.3 and 0.5/zg MK-801 groups were re-analyzed using only the latency scores that were below the median in each group. This analysis was based on the assumption that if two groups are comparable, the below-median retention latencies of one group should not differ from those of the other group. A Kruskal-Wallis ANOVA of the below-median retention latency scores of the vehicle, 0.2 and 0.5/zg MK-801 groups revealed significant differences among the three groups ( H 2 = 9.46, P <0.01). More importantly, Mann-Whitney U-tests based on the below-median retention latency data indicated that the 0.5 p,g MK-801 group differed significantly from the vehicle (U = 0, P < 0.01) and 0.2 /zg MK-801 (Uffi 1, P < 0.02) groups. Thus, the 0.5 /zg MK-801 group appears to consist of two heterogeneous populations, one with normal retention and the other with impaired retention. In an attempt to assess the validity of this analysis based on below-median retention latencies, the retention results of the D,L-APS, D-AP5 and CPP experiments reported above were reanalyzed by using only the below-median retention latency scores. For all three experiments this analysis (based on Kruskal-Wallis ANOVAs and Mann-Whitney U-tests) yielded results highly comparable to those based on all animals in each group.

Experiment H Since the N M D A antagonists were injected before training in Expt. I, the treatments might have affected performance in the CMIA task by altering processes not directly related to learning and memory. To address the possibility that the treatments may have altered sensitivity to the footshock punishment, rats were tested for footshock sensitivity 3 min after intraamygdala injections of the vehicle (n = 7) or D,L-AP5 (3 /~g, n = 9 or 10 /zg, n =9). The rats used in the second replication of D,L-AP5 experiment described in Expt. I above were tested in this experiment. Five days were allowed between the two experiments and the drug treatments were counterbalanced. There was no

40 effect of prior treatments used in Expt. I on the rats' responses in Expt. II. For the repeated-measures effect of footshock level, the non-parametric Friedman test 37 was performed on the reaction scores of each group: as shown in Fig. 5, the footshock level significantly affected the reaction scores in the vehicle (Xr2s= 26.631, P < 0.001), 3 /zg (Xr~- 36.481, P <0.001) and 10 /zg D,L-AP5 (Xr25= 34.737, P < 0.001) groups. However, ~,2 tests of the reaction scores obtained at each footshock level indicated that the vehicle and drug groups did not differ in reponsiveness at any footshock level (X42= 2.679, 2.797, 5.316, 4.526 and 1.277 for 0.1, 0.15, 0.2, 0.25 and 0.3 mA, respectively, P > 0.25 for all). At the footshock level of 0.35 mA which is considerably lower than that used in Expt. I, all animals reacted to the footshock with a score of 2.

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Fig. 6. Effects of intra-amygdala injections of D,L-AP5 on locomotor activity in Y-maze in Expt. Ill. There is no significant treatment effect.

Experiment !!! The effects of NMDA antagonists on locomotor activity were examined using a Y-maze as described in Materials and Methods. On day 1, animals were habituated to the maze. On day 2, baseline locomotor activity was measured. On day 3, vehicle (n--15), 3 (n = 13) or 10/zg (n = 15) D,L-AP5 was injected into the amygdala 3 min before testing, and the number of sections of the maze entered during 3 consecutive 2-rain sessions and the percent spontaneous alternation were recorded. Rats from the second and third replications of o-AP5 experiment described in Expt, ! above were used in this exneriment. The drug treatments were counterbalanced and 2-5 days were allowed between Expt, i and 111. There was no effect of prior treatments used in Expt. 1 on the rats' performance in Expt. I!1,

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0.15 0.2 0.25 0.3 FOOTSHOCK INTENSIFY(mA)

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Fig, 5. Effects o~" intra-amygdala injections of D,L-AP5 on footshock sensitivity in Expt, li, There is no significant drug effect,

When initially placed in the Y-maze on day 1, the animals were relatively immobile for 1-3 min. By day 3, the animals readily moved around the maze. One rat in the vehicle group was excluded from statistical analysis because it displayed emotional reactions (vocalization) when placed in the Y-maze and exhibited very low locomotor activity. The three groups did not differ in baseline locomotor activity, i.e. the number of sections entered (NSE) on day 2 (F.,,40 < 1). A two-factor repeated measures ANOVA with the drug treatment as the between-subject and the time (three 2-min sessions) as the withinsubject variable was performed on the NSE data obtained on day 3. The repeated measures effect was highly significant (F2,H, = 28,546, P < 0,001), As sitown in Fig, 6, the locomotor activity was initially high but decreased during the testing period, Neither the drug treatment main effect nor the interaction effect was significant (F2,~o < 1 and F,~,so= 2,131, 0,05 < P < 0,1, respectively), Although there appeared to he a tendency toward increased locomotor activity in the first 2 min session in D,L-APS-treated rats (see Fig. 6), one-way ANOVA of the NSE data for that test session did not reveal significant difference among the groups (Fz,4o = 2,52, 0,05 < P < 0,1), The alternation data from three rats (one from each group) that made fewer than 15 choices during the test period were discarded since the number of responses was insufficient for calculation of percent spontaneous alternation. The groups did not differ in spontaneous alternation. The means :!: S.E.M. percent spontaneous alternation were 69.8 :!: 2.95, 64.4 + 3.38, and 66.4 + 3.05 for the vehicle, 3 and 10 /zg D,L-AP5 groups, respectively (F2,37 < 1).

41

Experiment IV

12

In Expt. I, the animals were trained under the influence of the drug and tested without the drug. Thus, the retention impairment found in Expt. 1 may have resulted from a state-dependent retrieval deficit rather than from disruption of memory storage processes 64. According to this hypothesis, intraamygdala injections of NMDA antagonists administered before training and testing should result in retention performance comparable to that of the controls, whereas administering different treatments (e.g. vehicle and o-AP5 or vice versa) before training and testing should result in impaired retention performance. This state-dependency hypothesis was tested in Expt. IV by injecting either vehicle (VH) or NMDA antagonists into the amygdala before both the CMIA training and testing sessions. Doses of 10/zg O,L-AP5 (A10), 1 /zg D-AP5 (A1), and 0.5 /zg MK-801 (MK) were used in Expt. IVa, IVb and IVc, respectively. Thus, there were four pre-training-pre-testing treatment groups in each experiment: VH-VH in = 14), VH-A10 (n = 12), A10VH (n = 13) and A10-A10 (n - 11) in Expt. IVa; VHVH (n -- 11), VH-AI in --9), A1-VH in --8) and A1AI ( n - 12) in Expt. IVb; VH-VH (n = 12), VH-MK (n--10), MK-VH i n - 12) and MK-MK (n = 16) in Expt. IVe. O,L-AP5 was injected 3 rain before the training and testing phases. The injections of o-AP5 and MK-801 were administered 10 rain before training and testing. Each of the three experiments was performed twice and the analyses were based on the pooled data. The data from one rat in the A I-AI group were excluded from the analyses because its retention latency (600 s) was three standard deviations (S.D. - 163) greater than the mean (~-91.3) of this group calculated with this rat included. The retention latencies of the remaining rats in this group ranged from 6 to 96. The acquisition performance was analyzed by twoway ANOVAs with the pre-training and pre-testing treatments as two factors. The training latencies were not significantly affected by the pre-training intraamygdala injections of 10/zg O,L-AP5 (F~,4~, < 1), 1 p,g o-AP5 iFLas < 1), or 0.5/zg MK-801 (F1,46 = 3.27, 0.05 < P < 0.08). As shown in F~g. 7, the NTC was significantly affected by the pre-training intra-amygdala injections of 10/zg O,L-AP5 (Ft,46 "- 5.198, P < 0.03). However, individual t-tests revealed no difference between the groups ialthough the difference between the NTCs of the VH-VH and A10-VH groups approached significance, t25 = 1.99, 0.05 < P < 0.06). The doses of 1 ~g D-AP5 and 0.5 p,g MK-801 did not affect the NTC (Fi.35 < 1 and Fi,46 = 1.64, P > 0.2, respectively).

10

A

8

g

2 o VH-VH

m

VH-A10 A10-VH D,L-AP5 (10 ug)

A10-A10

12 _1

10

m IM.

8

O

6

W m

4

Z

2 0

LU

Z

mmmm VH-VH

12.

VH-A1 A1-VH D-AP5 (1 ug)

A1-A1

10. 864.

2. O.

mmWm VH-VH

VH-MK MK-VH MK-801 (0.5 ug)

MK-MK

Fig, 7, Effects of pre-training intra-amygdala injections of 10 /zg t),t,-AP5 (A), ! /zg I~-AP5 (B), or 0,5 p,g MK-8(}I (C) on the number of trials to criterion in the CMIA training in Expt. IV. Set: text for the abbreviations.

The retention latencies are shown in Fig. 8. Kruskal-Wallis one-way ANOVAs revealed that the retention latencies of the tbur groups differed significantly in Expt. IVa (Ha ffi 14.584, P < 0.003), IVb (H~ = 16.349, P < 0.005), and IVc (H 3 --- 12.049, P < 0.008). Mann-Whitney U-tests of the retention latency data revealed that the VH-VH group in Expt. IVa differed significantly from the VH.AI0 ( U = 36, P < 0.02), AI0-VH (U ffi 17, P < 0.001), and AI0-A10 (U = 33, P < 0.02) groups; the VH-VH group in Expt. IVb differed from the A1-VH (U - 5.5, P < 0.005) and AI-AI (U = 15, P < 0.005) groups; the VH-VH group in Expt. IVc differed from the MK-MK ( U - 35, P < 0.005) group. Also, there were significant differences between the VH-A1 and A1-VH groups (U = 6.5, P < 0.005) and between the VH-MK and MK-MK groups ( U 33.5, P < 0.02). These results indicate that induction of the 'same states' during the CMIA training and testing (i.e. in the A10-A10, A1-A1 and MK-MK groups) did not reinstate memory.

42

Experiment V Since the drugs in Expt. I were injected through guide cannulae implanted in the striatum terminating just above the amygdala, it is possible that the drug diffused dorsally, outside the cannulae 63, and affected the striatum which has high concentrations of NMDA receptors =7"-~.Therefore, in Expt. V, vehicle (n = 9) or 1 pg o-AP5 (n = 9) was injected into the striatum above the amygdala 10 min before training in the CMIA task. As shown in Fig. 1B, all of the injection needle tips terminated well above the amygdala in the internal capsule, giobus pallidus, or caudate putamen. One rat in the 1 pg D-AP5 group was excluded from analysis because it did not reach the acquisition criterion after receiving 14 training trials. This score was three standard deviations (S.D. = 3.72) from the mean (~ = 3.5) calculated with the data from this rat included. All other rats in that group required only 2 or 3

600,

A

500, 400, 300, 200,

a

a

a

100,

1

0 VH.VH

l

VH.A10 A10-VH rJ,L.APS (10 ug)

A10,A10

600 500

i m

400

ill

A

c

B

~00 T 500'

o

i 400'

! 300' -~ 200' O I too, 9

VEH

O-AP5 (1ug)

:8

VEH

D..AP5 (1ug)

o VEH

O-AP5

(1ug)

Fig. 9. Effects of pre-training intra-striatum injections of 1 g g D-AP5 on the training latency (A), number of trials to criterion (B), and retention latency (C) in the CMIA task in Expt. V. * Different from the vehicle group. See text for P values.

trials and those in the vehicle group required 1-3 trials. As shown in Fig. 9, in comparison with the vehicle controls, the 1 /zg D-AP5 group had shorter training iatencies (Ft.~-6.25, P<0.03) and higher NTCs (FLtt,= 5.333, P < 0.04). However, the retention latencies of the two groups did not differ significantly ( U 22.5, 0.05 < P < 0.09), although a significant drug effect on retention might have been masked by the ceiling effect. These findings indicate that injections of D-AP5 into the striatal area above the amygdala impaired acquisition, but not retention, of the CMIA response. This pattern of results shows sharp contrast with that of Expt. ! in which NMDA antagonists injected into the amygdala impaired retention, but not acquisition, of the CMIA response. Thus, it seems unlikely that the effects obtained in Expt. i were due to diffusion of the drug up along the guide cannulae to the striatum.

'300 200 100,

a, b

a

A1-VH

A1-A1

0 VH-VH

==

o w

VH-A1

D-APS (1 ug) 600.

0O'

I

400,

C

ool I'°°o ooi vtr VH-VH

I

VH-MK MK-VH MK-MK MK-801 (0.S ug) Fig. 8. Effects of p r e - t r a i n i n g / p r c . t e s t i n g intra-amygdala injections of Ill pg l),=-AP5 (A), I pg D-AP5 (B), or 0,5 pg MK-801 (C) on the rctemitm latency in Expt, IV, a, different from the VH-VH group; b, different from the VH-AI group: c, different from the VH-MK group, See text for P values,

Experiment VI If activation of amygdala NMDA receptors during training is essential for the subsequent retention of the CMIA response, blockade of the receptors should be expected to impair retention regardless of the amount of training given. However, if other neurotransmitter systems in the amygdala or brain areas other than the amygdala can compensate for the dysfunction of amygdala NMDA receptors, retention impairment induced by amygdala NMDA receptor blockade should be attenuated by giving additional training. Experiment VI addressed this issue. The rats in the VH-VH ( n - 6) and AI-AI ( n - - 6 ) groups of the second replication of Expt. IVb and the VH-VH (n = 12) and MK-MK (n = 16) groups of both replications of Expt. IVc were given additional training immediately after the first retention test as described in Materials and Methods (see Table I).

43 On day 1, neither the training latencies nor the NTC were significantly affected by intra-amygdala injections of 1 ~g D-AP5 ( F e e = 1.196, P > 0.25 and F e e = 2.551, P > 0.14, respectively) or 0.5/.tg MK-801 (FL26 = 1.804, P > 0.15 and Ft26 - 1.622, P > 0.2, respectively). (Note that these acquisition data and the first retention latency data reported below are part of those reported in Expt. IV.) As is shown in Fig. 10, on day 3, the number of re-learning trials to the 100 s acquisition criterion of the A1-A1 group did not differ from that of the vehicle controls (Fl,lO -- 1, P > 0.3). In contrast, the number of re-learning trials of the MK-MK group was significantly higher than that of the vehicle controis (FL:,~ - 5.812, P < 0.05). Paired t-tests revealed that significantly fewer trials were required for relearning on day 3 than for the original CMIA learning on day 1 in the A1-A1 (t s = 2.712, P < 0.05) and control group (VH-VH, t.s ffi 4.568, P < 0.01) and in the MK-MK (t~ -- 3.901, P < 0.005) and control group (VH-VH, t ~s ffi 3.574, P < 0.005).

[ ] Mean Training Latency (Day 1) [ ] Median (IQ) First Retention Latency (Day 3) [ ] Median (IQ) Second Retention Latency (Day 5) "6"

600.

A

~

500.

w

400.

>.

.,J

'I-

300.

0I T '

2OO100 -

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500 400

¢~ 300 [ ] INITIAL LEARNING ON DAY 1 • RELEARNING ON DAY 3

m~ 200 100 ILl

0 VH-VH

MK-MK MK-e01 (0.5 ug)

2

1

0 A1-A1

VH-VH

O.AP5 (1 ug)

Fig. il. Effects of pre.training intra-amygdala injections of ! /~g D-AP5 (A) or 0.5 #g MK.801 (B) on the training latency and the first and second retention latencies of the animals given additional training in Expt. VI. The initial training (day 1) and first retention test/additional training (day 3), but not the second retention tes! (day 5), were given after drug injections, a, different from their own second retention latencies; h, different from the second retention latencies el the VH-VH group; c, different from the first retention latencies of the VH-VH group. See text for P values.

4

3

/ =E 2

I~ii;~:~:~~.~ 1

:

VH-VH

MK-MK

MK-O01 (0.5 ug) Fig. 10. Effects of intra-amygdala injectio,:s of 1 ~g D-AP5 (A) or 0.5 ~g MK-801 (B) on the number of trials to criterion in the initial CMIA training on day 1 and in re-learning on day 3 in Expt. VI. The drugs were injected before training on both days I and 3. a, different from its own initial NTC on day 1; b, different from the number of re-learning trials of the VH-VH group on day 3. See text for P values.

As is shown in Fig. l lA, on day 3, the difference between the first retention latencies of the VH-VH and A1-A1 groups fell short of significance. (Note the small n's per group (n - 6); U - 35, 0.05 < P < 0.06.) On day 5, the second retention latencies of the A1-A1 group were significantly lower than those of the VH-VH group (U = 6, P < 0.05). More importantly, Wilcoxon test for two matched samples revealed that in the VH-VH and A1-A1 groups, the second retention latencies were significantly higher than the first retention latencies (z = 2.023, P < 0.05 and z = 2.201, P < 0.03, respectively). As is shown in Fig. 11B, on day 3, the first retention latencies of the MK-MK group were significantly lower than those of the VH-VH group (U = 35, P < 0.005). On day 5, the second retention latencies of the two groups did not differ (U = 67, 0.05 < P < 0.08). This

44 lack of difference might be due to a ceiling effect since the median retention latencies of both groups reached the maximum level of 600 s. Wilcoxon test for two matched samples revealed that the second retention latencies were significantly higher than the first retention latencies in both the VI-I-VH (z = 2.366, P < 0.02) and MK-MK (z = 3.408, P < 0.001) groups.

DISCUSSION

Effects of atnygdala NMDA receptor blockade on acquisition and retoztion of the CMIA response In general, the findings of the present study indicate that intra-amygdala injections of NMDA antagonists prior to training did not significantly affect acquisition, but impaired retention, of the CMIA response. That is, although the rats given NMDA antagonist injections into the amygdala learned the task rather normally, they did not ret~,in the memory as well as did the controls. Control experiments indicated that these effects are not likely to be due to the drug influences on the rats' sensory-motor ability, drug-induced state-dependency, or diffusion of the drug to other brain areas. The relative potencies of the competitive NMDA antagonists that induced significant effects on retention in the present study are consistent with physiological and pharmacological findings4'"".:'~.'~+','~N:I~-AP5 was approximately 2-3-times more potent than I),L-AP5, and CPP was 3-10-times more potent than o-AP5 in having significant effects on retention in Expt l. This relative potency relationship among the three drugs strongly suggests that the memory-impairing effects of intra-amygdala injections of these drugs were mediated through blockade of NMDA receptors. Also, the t),LAP5 doses found to impair retention in Expt. ! (3 and 10 ~tg, or 15.23 and 50.76 nmol, respectively) correspond well with those found to be effective in impairing Pavlovian fear conditioning (12.5-50 nmol)~7 and retention of a one-trial IA response (5 /zg) 4~ when administered into the basolateral amygdala. The effects of the non-competitive NMDA antagonist, MK-801, on retention were not comparable to those of competitive NMDA antagonists. The 0,5 /zg MK-801 group appeared to consist of two subgroups, one with normal memory and the other with impaired memnrv. The bimoda; retention performance of this MK-,~01 group obtained in Expt, ! was not replicated in Yxpt. IV. The basis of this discrepancy is not clear, alth(;ugh there is a procedural difference between the two experiments in that, unlike the 0.5 /zg MK-801 group in Expt. I, the MK-VH and MK-MK groups in

Expt. IV received intra-amygdala injections before testing. The present finding of different effects of competitive vs. non-competitive NMDA antagonists is consistent with the results of many other behavioral studies reporting differences between the actions of the two types of drugs 26'34'52'77'78'80'81. In Expt. IV, intra-amygdala injections of 10 p,g D,L-AP5 given 3 min before the retention test impaired retention performance: the retention latencies of the VH-A10 group were lower than those of the VH-VH group. Similar effects were found in pilot studies in which intra-amygdala injections of 3 ~g O,L-AP5 or 1 ~tg o-AP5 were given 3 rain before training and testing. These results may suggest a role of amygdala NMDA receptors in memory retrieval processes. However, the retention-impairing effects of pretesting NMDA antagonist injections were not replicated when o-AP5 or MK-801 was administered 10 min before testing. Thus, considered together with the evidence that high doses or acute injections of D,L-AP5 can cause depression of normal synaptic transmission in the hippocampUS15`lt''33'3')'4H'~'2,these findings suggest that the retention impairment induced by pre-testing NMDA antagonist injections into the amygdala may be due to side effects of the drugs (i.e. depression of general neuronal activity) rather than to blockade of NMDA receptors. if it is the case that the neuronal activity of the amygdala is depressed by D,L-AP5 or D-AP5 injected 3 rain before testing, it is possible that the retention impairment found in Expt. ! may have been due to non-specific inactivation of the amygdala during acquisition of the task rather than to specific blockade of amygdala NMDA receptors. However, this view is not supported by other findings: unlike the effects of 10 ~g D,L-AP5 given 3 rain before retention test, 1 ~g D-AP5 or 0.5 ~g MK-801 injections given 10 rain before the test did not impair retention performance in Expt. IV (see the VH-AI and VH-MK groups in Fig, 8B and C). Thus, these findings suggest that, when intra-amygdala injections of NMDA antagonists are administered 10 rain prior to training, their retention-impairing effects are not due to non-specific effects on acquisition.

Effects of the degree of training There are two main findings of Expt. Vi. First, in both the vehicle controls and the animals given NMDA antagonist injections, fewer trials were required for re-learning (day 3) than for the original learning (day 1) of the CMIA. Importantly, even though the animals given NMDA antagonists showed impaired performance on the first retention test on day 3, they showed savings in re-learning the CMIA task that day. This finding indicates that the animals were not completely

45 amnestic. Second, additional training significantly improved the retention performance of the animals given NMDA antagonists as well as the controls: in both groups, the second retention latencies were significantly higher than the first retention latencies. Thus, the retention-impairing effects of amygdala NMDA receptor blockade were attenuated by giving additional training to the animals. This result is consistent with previous findings indicating that the effects of amygdala lesions may vary depending on the degree of original, training 6'zs'4°'76. There are several possibilites that may account for the effects of additional training on the CMIA retention. First, the drugs may not have blocked all NMDA receptors in the amygdala and the remaining unblocked receptors might have been activated by additional training. However, there is evidence suggesting that most of the amygdala may have been affected by the drugs: Morris et al.62 found that a 1/zl injection of 10 mM [3H]D,L-AP7 (i.e. 2.252 ~g), a compound structurally similar to AP5, into the rat hippocampus spread more than 5 mm medio-laterally and 4 mm rostrocaudally. The main part of the rat amygdala, including the central, lateral, and basolateral nuclei, is approximately 2.5 mm long, 2 mm high at its thickest part, and 1.8 mm wide at its widest part (estimation based on Paxinos and Watson6~). In spite of many differences between the Morris et al. study ¢'2 and the present study, such as the injection volume and structures involved, this comparison suggests that it is possible that most, if not all, of the amygdala NMDA receptors were affected by the NMDA antagonists administered in 0,5/zl volume, Second, the doses of NMDA antagonists used (1/zg D-AP5 and 0.5/zg MK-801) in Expt. VI may not have been high enough to exert strong blocking effects on NMDA receptors. However, other evidence indicates that a 1 /zg dose of D-AP5 blocks the memory-improving effects of a moderate amount of additional training: in the first replication of Expt. IVb, animals in the VH-VH and A1-A1 group received re-learning trials (see Table l) but not escape training trials on day 3 immediately after the first retention test. On the second retention test on day 5, the VH-VH group showed significantly improved retention performance whereas the AI-A1 group did not (z =2.023, P<0.05 and z = 1.461, P > 0.1, respectively, Wilcoxon test for two matched groups). These results, together with the findings of Expt. VI, suggest that the retention-impairing effects of NMDA antagonists injected into the amygdala depend on the degree of training. A third possibility is that the competitive blockade of NMDA receptors may have been overcome by in-

creased release of glutamate/aspartate induced by additional training. However, the finding that the memory-impairing effects of intra-amygdala injections of the non-competitive antagonist, MK-801, were also attenuated by additional training provides evidence against this suggestion, although it remains possible that the dose of MK-801 used (0.5 p.g) was not high enough to block all available NMDA-gated ion channels. The bimodai retention performance of the 0.5 /zg MK-801 group found in Expt. I, although not replicated in Expt. IV, suggests the fourth possibility. That is, the improved performance of the MK-MK group on the second retention test may have been due to good retention performance of some rats with above-median retention latencies (see Fig. 4D). Conversely, the rats in Expt. VI that showed below-median retention latencies on the first retention test may not have improved even after receiving additional training. However, reanalysis of the below-median retention latency data according to the method described in Expt. I revealed that these rats showed significantly improved retention performance on the second retention test (z = 2,521, P < 0.02, Wilcoxoa test for two matched samples). Lastly, NMDA receptors in the amygdala may not be solely responsible for the CMIA retention. Rather, other neurotransmitter systems in the amygdala or other brain areas may participate in long-term retention of the response.

Theoretical implications The main findings of this study can be summarized as follows: blockade of amygdala NMDA receptors induced prior to training does not significantly affect acquisition, but impairs retention, of the CMIA response, and this retention-impairing effect can be attenuated by giving additional training to the animals. The present finding of retention impairment induced by amygdala NMDA receptor blockade is consistent with the general findings of many studies examining the effects of either systemic or central injections of NMDA antagonists on retention of one-trial IA response 1&1~,22,3°'49'65'7'~.However, the finding that NMDA antagonists did not affect acquisition contrasts with the those of other studies indicating that NMDA antagonist injections, either central or peripheral, prior to training disrupt acquisition of spatial memory in Morris water maze or radial arm maze x~'~'3a'o°'¢'9'72.This difference suggests that the role of NMDA receptors in acquisition and retention of a learned response may depend on the type of tasks and/or the brain areas involved. In particular, the present findings strongly suggest that NMDA receptors in the amygdala are

46

involved primarily in long-term retention rather than in acquisition of an IA response, Long-term potentiation (LTP), the long-lasting increase of synaptic efficacy induced by brief tetanic stimulation of afferent pathways "~'4~',is hypothesized by many investigators to be a neurobiological mechanism underlying long-term information storage in the mammalian brain 13"14'53''~4"t'1'74'75. Recent experiments demonstrated that LTP can be induced in the amygdala in vitro ~° and in vivo 2~. The drugs used in the present study have been shown to block the induction of LTP in the hippocampal areas z'z2.lS'31'3"~'6s. If amygdaloid LTP is dependent on NMDA receptor activation, the present findings suggest that LTP within the amygdala may be involved in retention of the CMIA response. From this perspective, our findings fit well with the view that the amygdala is a locus of changes underlying the formation of stimulus-reinforcement associat ions 2.27.42.47.73. However, it is not clear that the effects of NMDA antagonists found in this study are due to blocking of LTP. It may be that amygdaloid LTP is not dependent on NMDA receptor activation. In addition, there is evidence that NMDA antagonists such'as APS, CPP, or phencyclidine antagonize NMDA-induced release of acetylcholine and dopamine in the nucleus accumbens 44 and NMDA-induced release of norepinephrine in the hippocampus 4'~'7~. Also, systemic injections of MK-801 increases dopamine metabolism in the amygdala a~d other brain areas t'7. Taken together with the evidence that many neurotransmitter systems in the amygdala arc involved in memory consolidation processes ~,.~¢', these findings suggest that the effects of intra-amygdala injections of NMDA antagonists found in the present study may have been mediated through interaction of the NMDA receptors with other neurotransmitter systems in the amygdala. For example, the blockade of NMDA receptors might have inhibited noradrenergic functioning in the amygdala (K.C. Liang, personal communication) which is known to be involved in modulating inhibitory avoidance learning :~,s°,-~'. Extensive evidence indicates that post-training manipulations of the amygdala affect subsequent retention in a time-dependent manner 5°.55,5~'. This and other evidence has suggested that the amygdala serves a role in modulating the consolidation of long-term memory occurring in other brain areas after learning ~a.Ss. The present finding indicating that amygdala NMDA receptor blockade did not affect the acquisition of the CMIA response fits well with this view: as modulation of memory storage presumably occurs after learning, d~sfunction of the amygdala during learning would not be expected to affect acquisition performance. The

finding that additional training improved retention in spite of amygdala NMDA receptor blockade is also consistent with this view: without the modulatory influence from the amygdala, memory storage in other brain areas can still occur, although it will be weaker. In animals given intra-amygda!a injections of NMDA antagonists, additional training would be expected to facilitate memory storage by compensating for the lack of modulatory influence from the amygdala. From this viewpoint, the finding of retention impairment induced by amygdala NMDA receptor blockade suggests that the NMDA receptor system in the amygdala is one of several neurotransmitter systems involved in modulating long-term memory storage in other brain regions. Acknowledgements. This research was supported by U PHS Grant

MH 12526 from NIMH and NIDA, and ONR Contract N00014-J1626. We thank M. Campeau, W. Falls, and M.J.D. Miserendinofor commenting on this paper and N. Collett for assistance in the preparation of the manuscript. REFERENCES Abraham, W.C. and Mason, S.E., Effects of the NMDA receptor/channel antagonists,CPP and MKg01, on hippocampal field potentials and long-term potentiation in anesthetized rats, Brain Res., 462 (1988) 40-46. Aggleton, J.P. and Mishkin, M., The amygdala: sensory gateway to the emotions, in R. Plutchik and H. Kellerman (Eds.), Emo. tion: Theory, Research, and Experienct; VoL 3, Academic Press, London, 1986, pp. 281-299. Alessandri, B., Battig, K. and Welzl, H., Effects of ketamine on tunnel maze and water maze performance in the rat, Behat,,

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