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Strain-dependent effects of γ-l -glutamyl-l -aspartate, a NMDA antagonist, on retention of a Y-maze avoidance learning task in mice
Behavioural Brain Research, 55 (1993) 69-75 © 1993 Elsevier Science Publishers B.V. All rights reserved. 0166-4328/93/$06.00
69
BBR 01438
Strain-dependent effects of y-L-glutamyl-L-aspartate, a N M D A antagonist, on retention of a Y-maze avoidance learning task in mice A r i e l l e U n g e r e r '~, C l a u d i n e M r l a n a a n d J e a n D e B a r r y b a Laboratoire de Psychophysiologie, Universit~ Louis Pasteur, URA 1295 CNRS, Strasbourg (France) and b Centre de Neurochimie, CNRS, Strasbourg (France)
(Received 7 April 1992) (Revised version received 10 December 1992) (Accepted 4 February 1993) Key words: 7-L-Glutamyl-L-aspartate; Memory; Active avoidance; Inbred strain; N-Methyl-D-aspartate receptor; Mouse
The NMDA receptor antagonist, 7-L-glutamyl-L-aspartate (7-LGLA), suppressed spontaneous improvement in posttraining performance in Swiss mice during the hours following acquisition of a Y-maze avoidance learning task. Since variability in posttraining performance is at least partially due to genetic factors, we compared the effects of 7-LGLA on retention of Y-maze learning in C57BL/6J, DBA/2J and BALB/c mice. Mice had to leave the start alley of the maze within the first 5 s (temporal component) and to choose the left alley (spatial component). C57BL mice significantly improved their performance from 1 h to 24 h posttraining, whereas DBA/2J and BALB/c mice did not. However, only retention of the temporal component improved over time in C57BL. 7-LGLA administered immediately posttraining (0.025-25 #mol/kg, i.p.) dose-dependently impaired retention of the temporal component in C57BL mice 48 h later, but had no significant effect on retention of the spatial component. ~,-LGLA administered 24 h posttraining induced a similar but weaker deficit. In contrast, ~,-LGLA did not significantly affect retention of DBA/2J and BALB/c mice, regardless of the component analyzed or the time of administration. It had no effect on locomotor activity or emotional reactivity of animals of any strain. These results support the hypothesis of a specific action of y-LGLA on mechanisms involved in the treatment of information during the hours following acquisition, and suggest that NMDA receptors are involved in this action.
INTRODUCTION
Convergent data demonstrate that N-methyl-Daspartate ( N M D A ) receptors are critically involved in the induction of long-term potentiation (LTP) of synaptic responses at the hippocampal and cortical levels 1'3'9, suggesting that they may have a central role in learning and memory processes 13'24. This hypothesis originally received support from studies by Morris et al. ~7 who showed that the specific and competitive N M D A receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5) chronically infused in the lateral ventricle (ICV) blocked the induction of LTP and strongly disrupted acquisition of a spatial learning task in a water maze; D-AP5 did not affect acquisition per se, short-term memory or retrieval processes, but appeared to interfere specifically with formation of memory traces, which agrees with the hypothesis. In contrast, D-AP5 administered in the same conditions had no Correspondence: A. Ungerer, Laboratoire de Psychophysiologie, Universit6 Louis Pasteur, URA 1295 CNRS, 7 rue de l'Universit+, 67000-Strasbourg, France. Fax: (33) 88 35 84 49.
effect on acquisition of a visual discrimination in the water maze, suggesting a highly task-specific action 17. Task- and time-dependent effects of N M D A antagonists were subsequently confirmed in various learning tasks. For example, ICV infusion of D-AP5 retarded acquisition of an olfactory discrimination task, but had no effect on acquisition or retention of a one-way active avoidance learning z3. Elsewhere, pretraining administration of CGS 19755 (cis-4-phosphonomethyl-2piperidine carboxylate), a potent competitive N M D A antagonist, induced a marked retention deficit in a stepthrough passive avoidance task, but did not affect retention of a step-down passive avoidance learning ~2. Moreover, the noncompetitive N M D A antagonist, MK-801 [( + )-5-methyl- 10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5,10-imine maleate], impaired retention of a step-through dark avoidance learning task when administered prior to training and improved it when administered immediately posttraining, whereas both pre- and posttraining administration of MK-801 improved retention of a step-down passive avoidance task tr. Previous work suggests that N M D A receptors might
70 be specifically involved in mechanisms underlying longterm consolidation of memory traces, as indicated by behavioral effects of N M D A antagonists in a Y-maze avoidance learning task in which animals had to leave the start alley of the maze within the first 5 s (temporal component) and to choose the left alley (spatial component) to avoid foot-shock. Indeed, we showed that systemic posttraining administration of 7-L-glutamyl-L-aspartate (7-LGLA), a pseudo-peptide which has the parmacological properties of a competitive N M D A antagonist 14"26, specifically suppressed the spontaneous improvement in performance observed in control animals during the hours following acquisition27; 7-LGLA significantly impaired retention of the temporal component of the task, which improved significantly over time, whereas it had no effect on retention of the spatial component, which did not improve over time; moreover, ),-LGLA did not affect spatial recognition memory in an alternation task in which control animals showed no posttraining improvement in performance 25. Similar behavioral effects were observed following systemic administration of CPP ([3(2(carboxypiperazin-4-yl)propyl- 1-phosphonate], a potent and specific competitive N M D A antagonist, or ICV administration of D-AP5, suggesting a specific action of N M D A antagonists on mechanisms underlying spontaneous improvement in posttraining performa n c e 15,26.
Variability in posttraining performance over time is known to be at least partially due to genetic factors. The purpose of the present study, in light of these data, was to compare the effects of 7-LGLA on retention of a Y-maze avoidance learning task in three strains of mice, C57BL/6J, BALB/c and DBA/2J, which differ in mnemonic capacities and in posttraining evolution of performance 2'4't°'2°. A control experiment was also carried out using an open-field test procedure to determine whether memory deficits induced by ? - L G L A might be due to an action on locomotor and/or emotional reactivity of the animals. MATERIALS AND M E T H O D S
Animals Male mice of C57BL/6J, BALB/c, and DBA/2J strains, 6 weeks old, were purchased from IFFA-Credo (69210-1'Arbresle, France) and served as subjects. After arriving at the laboratory, they were housed under standard conditions on a 12:12-h light-dark cycle (lights on at 7.00 a.m. and off at 7.00 p.m.) with food and water ad libitum. The experiment started when they were 2 months old and weighed 25-35 g . Behavioral testing was carried out during the light phase of the cycle.
Y-maze avoidance learning task Mice were trained in a transparent Plexiglas Y-maze with three alleys (each 13 x4.5 × 5.5 cm) set on the median lines of an equilateral triangle. At the end of each alley was a transparent Plexiglas mobile box ( 1 0 x 4 . 5 x 5.5 cm) with an opaque Plexiglas sliding door which allowed transport of the animal from the goal alley to the start alley without handling. At the start of each trial, the animal entered the start alley from the mobile box and underwent a double conditioning procedure in which it had to leave the start alley within the first 5 s (temporal component) and to choose the left alley of the maze (spatial component) in order to avoid footshock (35 V AC, 4.5 mA; duration 1 s every 5 s). The animal was held to be making an avoidance error when it failed to leave the start alley within 5 s, a discrimination error when it entered the fight alley at least once, and an incorrect trial when it made either an avoidance error, or a discrimination error, or both. The animal underwent one trial every minute until it reached a criterion of 7 correct out of 8 consecutive trials. It was submitted to a second learning session at various delays following the first session in order to test retention. The number of incorrect trials, avoidance errors and discrimination errors made during the first and the second sessions were recorded. Statistical analyses used a two-factor ANOVA. Significant differences among groups were analyzed by the Newman-Keuls procedure. The Kruskal-Wallis test was used for analyses of discrimination errors during retention testing as their distribution was not normal in some groups. In the first experiment, four groups of mice of each strain were submitted to a first learning session and, 1 h, 3 h, 6 h, or 24 h later, to a second learning session in order to test retention. Each subject was tested only once. In the second experiment, immediately after the learning session mice of each strain received an intraperitoneal (i.p.) administration of 0.025, 0.25, 2.5 or 25 #mol/kg ?-LGLA dissolved in 0.9°Jo NaC1 (25 ml/kg). Control mice received 25 ml/kg 0.9°o NaC1. Learning retention was tested 48 h following the pharmacological treatment. In the third experiment, 7-LGLA (0.25, 2.5, 25 #mol/kg, i.p.) or vehicle was administered 24 h after the learning session and retention testing was carried out 48 h later. In the three experiments each group consisted of 11-12 mice. Exploratory activity in an open,field Behavioral effects of 7-LGLA on exploratory activity of C57BL/6J, BALB/c and DBA/2J mice were analyzed in a square open-field (50 × 50 x 50 cm) divided into 25 equal squares and including a central platform (8 x 8 x 4 cm) and 4 small pillars (2 × 2 × 6 cm) each set
71 at 10 cm from the two adjacent walls of the apparatus. Mice received an administration of either ~,-LGLA (0.25 or 25/~mol/kg, i.p.) or 0.9% NaC1 and 30 min later were placed in the apparatus for 30 min. The number of square crossings, rearings, crossings of the central platform and fecal boluses were recorded in every block of 5 min. Each group of mice contained 16 subjects. Data were analyzed by a two-factor ANOVA.
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Y-maze avoidance learning task Strain differences in acquisition and retention. Performance expressed by the number of incorrect trials made by C57BL/6J, BALB/c and DBA/2J mice during the first learning session and during retention testing carried out 1 h, 3 h, 6 h, or 24 h posttraining are shown in Fig. 1. Mice of the three strains showed significant differences in acquisition of the task (F2a2o=21.85; P<0.001) during the first learning session: DBA/2J mice made significantly fewer incorrect trials than BALB/c and C57BL mice, which did not differ from each other (Newman-Keuls test: DBA/2J vs. BALB/c P<0.01; DBA/2J vs. C57BL, P<0.01; BALB/c vs. C57BL, P>0.05). Performance also differed between strains in terms of the number of avoidance errors (F2,120 18.68; P < 0.001 ) and discrimination errors (F (2,120) -- 9.65; P < 0.001) (Fig. 2): DBA/2J mice made fewer avoidance errors than C57BL and BALB/c mice (Newman-Keuls test, P < 0.01), but C57BL made more avoidance errors than BALB/c mice ( P < 0.05); in contrast, C57BL mice did not make significantly more discrimination errors than DBA/2J mice, unlike BALB/c =
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Fig. 1. Mean ( _+S.E.M.) number of incorrect trials made by C57BL, DBA/2J and BALB/c mice during the learning session and during retention testing in a Y-maze avoidance learning task as a function of training-to-test interval.
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mice (Newman-Keuls test, BALB/c vs. DBA/2J, P<0.01; BALB/c vs. C57BL, P<0.05; DBA/2J vs. C57BL, P>0.05). No significant difference was observed during the learning session between the four groups of each strain (1 h, 3 h, 6 h or 24 h) either for the number of incorrect trials (F3,12o = 0.917; P > 0.05), or the number of avoidance (F3a20 = 0.96) or discrimination e r r o r s (/173,120 = 0.41). Retention performance, expressed by the number of incorrect trials during the second learning session (Fig. 1) differed markedly across strains (F2a20 = 13.18; P < 0.001) and according to the time of retention testing (F3a2o=3.97; p=0.009), but no significant strain x time interaction was observed (F6,12o= 1.93; P>0.05). Comparisons between strains showed that DBA/2J and BALB/c mice did not differ significantly at any posttraining time. In contrast, C57BL mice showed impaired performance compared to DBA/2J and BALB/c mice 1 h and 3 h posttraining ( N e w m a n Keuls test, P<0.01), but not 6 h or 24 h posttraining. Moreover, comparisons within each strain showed that
72 C57BL mice significantly improved their performance from the first to the 24th hour following acquisition (Newman-Keuls test: 1 h vs. 24 h, P<0.01; 3 h vs. 24 h, P<0.01; 6 h vs. 24 h, P<0.05), whereas DBA/2J and BALB/c mice did not exhibit a time-dependent improvement in posttraining performance. The same pattern of results was obtained for the number of avoidance errors, which differed both across strains (F2,12o = 27.90; P<0.001) and according to the testing time (F3,120 = 8.20; P<0.001) (Fig. 2A): C57BL mice made more avoidance errors than DBA/2J and BALB/c mice at 1 h, 3 h, and 6 h posttraining (Newman-Keuls test, at 1 h: P<0.01; at 3 h: C57BL vs. DBA/2J, P<0.01; C57BL vs. BALB/c, P<0.05; at 6 h: C57BL vs. DBA/2J, P<0.05; C57BL vs. BALB/c, P<0.01), but not 24 h posttraining, and they showed significant improvement in performance between the first and the 24th hour following training (Newman-Keuls test, P < 0.01). DBA/2J and BALB/c mice did not improve performance and did not differ from each other at any time. In contrast, no significant difference in the number of discrimination errors (Fig. 2B) was observed between groups (Kruskal-Wallis test, H = 8.18, P > 0.05), indicating that the three strains did not differ for this parameter whatever the time of retention testing.
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Fig. 3. Dose-dependent effects of 7-LGLA on retention of a Y-maze avoidance learning task in C57BL, DBA/2J and BALB/c mice, as expressed by the number of incorrect trials (mean +_S.E.M.) made during retention testing. The pharmacological treatment was administered immediately following the learning session and retention was tested 48 h later.
mice whatever the dose administered and the parameter considered. In contrast, it induced a dose'dependent {SSS] NaCI t-'/q 0.025 0.25 E;~ 2.50
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Effects of posttraining administration of 7-LGLA on retention.There was no difference in acquisition of the task among experimental groups within each strain during the first learning session prior to 7-LGLA administration, but a strong strain effect was found for the number of incorrect trials (F2,,62 = 9.47, P < 0.001), the number of avoidance errors (F= 18.38) and the number of discrimination errors (F=6.05, P<0.01), thus confirming the results of the first experiment. 7-LGLA administered immediately posttraining deeply impaired retention performance 48 h later, as expressed by the number of incorrect trials made by the animals during retention testing. This deficit was both strain and dose-dependent (strain effect: F2.162 = 8.25, P < 0.001; dose effect: F4,162 = 7.73, P<0.001; strain x dose interaction, F8,162 = 0.78, P>0.05) (Fig. 3), But 7-LGLA appeared to affect primarily retention of the temporal component of the task, as shown by the significant increase in the number of avoidance errors (strain effect: F2,16 z = 17.34, P<0,001; dose effect: F4,16 2 = 8.90, P<0,001; strain x dose interaction, Fsa62 = 1.15, P > 0.05) (Fig. 4A), whereas the number of discrimination errors did not differ between groups (KruskalWallis test, H = 10.31, P > 0.05) (Fig. 4B), indicating that retention of the spatial component was not impaired. However, comparisons of experimental groups within each strain showed that 7-LGLA had no significant effect on learning retention in DBA/2J or BALB/c
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Fig. 4. Dose-dependent effects of 7-LGLA on retention of the temporal component (A: avoidance errors) and spatial component (B: discriminationerrors) of a Y-maze avoidance l e ~ i n g task in C57B L, DBA/2J and BALB/c mice. 7'LGLA was administered immediately following the. learning session and retention was tested 48 h later.
73 retention deficit in C57BL mice treated at doses of 0.25 to 25 #mol/kg 7-LGLA, as reflected by a significant increase in the number of incorrect trials and of avoidance errors made by these animals compared to control mice (Newman-Keuls test, for the two parameters: 0.25 7-LGLA vs. NaCI, P<0.05; 2.5 and 25 7-LGLA vs. NaCI, P < 0.01) (Fig. 3 and 4A). But, even in C57BL, 7-LGLA had no effect on the number of discrimination errors whatever the dose administered (H=3.52, P > 0 . 0 5 ) (Fig. 4B). To test whether retention deficits observed in C57BL mice were due to motor side effects, mice of this strain were submitted to the Y-maze learning task in the same conditions as above, except that they had to leave the start alley of the maze within the first 10 s, instead of 5 s, at the start of each trial. As observed in the preceding experiment, C57BL mice treated at the dose of 2.5 #mol/kg 7-LGLA immediately posttraining made a greater number of incorrect trials (F,.2o = 4.40; P < 0.05) and of avoidance e r r o r s ( F t , 2 0 = 7.68; P<0.025) than control animals, but did not differ from them in the number of discrimination errors, thus indicating that retention deficits induced by 7-LGLA are unlikely related to an action on motor activity. 7-LGLA administered 24 h after the first learning session had little effect on learning retention 48 h after pharmacological treatment, as measured by the number of incorrect trials (dose effect, F3,m7 = 2.05, P > 0.05) or discrimination errors (Kruskal-Wallis test, H = 6.35, P > 0.05) by treated and control animals; but it induced a strain- and dose-dependent increase in the number of avoidance errors (strain effect, F2,x27 = 24.75, P < 0.001; dose effect, F3,m7 = 4.32, P = 0.006; strain × dose interaction, F6.127=1.06, P > 0 . 0 5 ) (Fig. 5), which was
mainly due to impaired performance in C57BL mice treated at the higher dose of 7-LGLA (Newman-Keuls test: 25 7-LGLA vs. NaC1, P < 0.05). The other experimental groups did not differ from each other, regardless of strain or dose administered.
Effects of 7-LGLA on exploratory activity Behavioral effects of 7-LGLA were analyzed in mice submitted to an open-field test in order to determine whether retention deficit induced by the drug could be due to an action on some behavioral components that might interfere with the animal's performance. Results showed that C57BL, BALB/c and DBA/2J mice exhibited strong strain differences in locomotor activity, as expressed by the number of squares crossed during the first 5 min (strain effect, F2,~3s = 20.05, P < 0.001) or the total duration of the test (strain effect, F 2 , 1 3 5 = 16.85, P<0.001), DBA/2J mice being significantly more active than BALB/c or C57BL mice (Newman-Keuls test, P < 0.01). But 7-LGLA, administered at doses of 0.25 or 25 #mol/kg, had no significant effect either during the 5 first min (dose effect, Fz,~3s = 0.334, P > 0.05; strain x dose interaction, F4,135 -- 1.01, P>0.05) or the total duration of the test (dose effect, F2,~35= 1.38, P>0.05; strain x dose interaction, Fa,t35= 0.75, P > 0.05) (Fig. 6). Likewise, 7-LGLA had no effect on the number of rearings, crossings of the central platform, or boluses, whatever the strain or the dose used. Moreover, 7-LGLA, when administered 48 h prior to open-field testing at the dose of 25 #mol/kg, did not affect performance of C57BL mice, as expressed by the number of squares crossed during the first 5 min of the test (treated group, M = 218.81 + 12.47; control group, M = 238.68 + 12.62) or the number of rearings (treated
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Fig. 5. Dose-dependent effects of ~,-LGLA on retention of the temporal component (avoidance errors: mean + S.E.M.) of a Y-maze avoidance learning task in C57BL, DBA/2J and BALB/c, when 7-LGLA was administered 24 h after the learning session and retention tested 48 h later.
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Fig. 6. Effects of 7-LGLA on locomotor activity of C57BL, DBA/2J and BALB/c mice in an open-field. Results are expressed by the number of squares crossed (mean + S.E.M.) during the 30 min of testing. 7-LGLA (0.25 or 25/~mol/kg, IP) was administered 30 min before the beginning of the test.
74 group, M = 26.31 + 9.10; control group, M = 23.18 + 8.33). Thus, retention deficits observed in this strain of mice do not appear to be due to non-specific proactive effects of 7-LGLA on performance.
DISCUSSION
Among the three strains of mice used in the present study, only C57BL mice displayed spontaneous improvement in posttraining performance over time, thus confirming that such an improvement is controlled by genetic factors as shown by others 2'4'1°'2°. BALB/c mice acquired the task at a rate similar to that of C57BL mice and slower than DBA/2J mice, as shown by the number of incorrect trials during the learning session, but they did not show posttraining improvement in performance, in contrast to C57BL mice, and did not differ in retention performance from DBA/2J mice whatever the time of testing. Moreover, mice of the three strains differed significantly in acquisition of both the temporal and the spatial components of the task, but did not show differences on retention of the spatial component, whereas retention of the temporal component significantly improved over time in C57BL mice but not in the two other strains. These results clearly show a dissociation between the two components of the task, as retention of the temporal component improved during the hours following acquisition in C57BL mice, in contrast to retention of the spatial component which did not, confirming observations using the same task with Swiss mice 25'26. Posttraining improvement in performance is currently thought to reflect an organization process of memory traces and would be related to low levels of training during original learning 5'7'8. In light of these data, it must be emphasized that C57BL mice made significantly more avoidance errors than BALB/c and DBA/2J mice and more avoidance errors than discrimination errors during the learning session (F1.8o = 26.37, P < 0.001). This suggests that the temporal component is more complex than the spatial one and is acquired less in C57BL mice than in DBA/2J and BALB/c mice, which would explain why retention of this component improves over time in C57BL mice. In this way, the lack of posttraining improvement in performance of DBA/2J and BALB/c mice would be due to an overlearning of the two components of the task and not to an incapacity of these strains to exhibit such a phenomenon. In agreement with this hypothesis, DBA/2J and BALB/c mice have been shown to display timedependent improvement in posttraining performance in a two-way avoidance learning task 2 and in a lever-press
conditioning task 4, respectively. Moreover, C57BL mice did not show such an improvement in this latter t a s k 4 o r in a visual discrimination learning task ~~. These data clearly demonstrate that posttraining improvement in performance is highly dependent on experimental conditions, in addition to genetic factors. 7-LGLA, when administered immediately posttraining, deeply impaired retention of the temporal component in C57BL mice. But it did not affect retention of the spatial component in C57BL mice and had no significant effect on retention of DBA/2J and BALB/c mice whatever the learning component considered. Also, 7-LGLA had no effect on locomotor activity or emotional reactivity of any of the animals in open-field testing, thus confirming its lack of toxicity at the doses used and indicating that retention impairment observed in C57BL mice is not due to an action on these behavioral components. In fact, the retention deficit induced by 7-LGLA appears highly correlated with posttraining improvement in performance displayed by C57BL mice on retention of the temporal component. Moreover, 7-LGLA, at doses of 2.5 or 25 #mol/kg, induced a deficit in retention of the temporal component which had the same amplitude as that observed in C57BL mice tested 1 h posttraining: thus, 3,-LGEA completely suppressed improvement in performance displayed by control mice between the first and the 24th hour following the learning session. However, ?,-LGLA still induced a weak deficit in retention of the temporal component when administered 24 h posttraining in C57BL mice, suggesting that organization of memory traces is not completely achieved by then. Indeed, retention performance of the temporal component slightly improved in C57BL control mice between 24 and 48 h posttraining, which agrees with the hypothesis. Moreover, delays for maximum improvement in performance have been shown to vary according to the learning task and the degree of acquisition and to frequently exceed 24 h (refs. 5, 6, 19, 22). The selective action of 7-LGLA on retention of the temporal component in C57BL mice is similar to that induced by ,/-LGLA, CPP or D-AP5 in the same task, but in Swiss mice 15"26. These data thus strengthen the hypothesis that N M D A receptor antagonists specifically interfere with mechanisms underlying posttraining improvement in performance and that N M D A receptors are involved in this action. However, the question is raised whether interstrain differences in ?,-LGLA effects reflect genetic differences in excitatory amino acid systems, and more specifically in N M D A receptors. Unfortunately, we lack relevant data. A preliminary study carried out by us on hippocampal membranes showed that 7-LGLA displaced [3HI-L-glutamate in
75 the three strains, but no significant difference was observed in site densities or dissociation constant between them. Elsewhere, Peterson et al. ~8 did not find any differences in density or affinity of N M D A receptors between C57BL and BALB/c adult mice on a whole brain membrane preparation. However, it cannot be excluded that such differences may exist, for example in some subfields of the hippocampal formation. Indeed, recent work 2~ showed that the size of the mossy fiber terminal fields varies across strains of mice, including C57BL, BALB/c and DBA/2J and is negatively correlated with performance in a spatial working memory task. In light of these data, interstrain studies on N M D A receptor pharmacology may be a valuable tool for a better understanding of these receptors and of their role in learning and memory processes.
ACKNOWLEDGEMENTS
This work was supported by D R E T (85/158, 88/ 057). We thank Dr. A. Mann for synthetizing ~-LGLA and Dr. J. Anderson for his help in carefully reading the manuscript.
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11 Kitahama, K., Valatx, J.-L. and Jouvet, M., Paradoxical sleep deprivation and performance of an active avoidance task: impairment in C57BR mice and no effect in C57BL/6 mice, Physiol. Behav., 27 (1981) 41-50. 12 Lehmann, J., Hutchison, A.J., Mc Pherson, S.E., Mondadori, C., Schmutz, M., Sinton, C.M., Tsai, C., Murphy, D.E., Steel, D.J., Williams, M., Cheney, D.L. and Wood, P.L., CGS 19755, a selective and competitive N-methyl-D-aspartate-type excitatory amino acid receptor antagonist, J. Pharmacol. Exp. Ther., 246 (1988) 65-75. 13 Lynch, G. and Baudry, M., The biochemistry of memory: a new and specific hypothesis, Science, 224 (1984) 1057-1063. 14 Mathis, C., De Barry, J. and Ungerer, A., NMDA antagonist properties of gamma-7-glutamyl-L-aspartate demonstrated on chemically-induced seizures in mice, Eur. J. Pharmacol., 185 (1990) 53-59. 15 Mathis, C., De Barry, J. and Ungerer, A., Memory deficits induced by -y-glutamyl-L-aspartate and D-2-amino-5-phosphonovalerate in a Y-maze avoidance learning task: relationship to NMDA receptor antagonism, Psychopharmacology, 105 (1991) 546-552. 16 Mondadori, C., Weiskrantz, L., Buerki, H., Petschke, F. and Fagg, G.E., NMDA receptor antagonists can enhance or impair learning performance in animals, Exp. Brain Res., 75 (1989) 449456. 17 Morris, R.G.M., Anderson, E., Lynch, G.S. and Baudry, M., Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5, Nature, 319 (1986) 774-776. 18 Peterson, C. and Cotman, C.W., Strain-dependent decrease in glutamate binding to the N-methyl-D-aspartic acid receptor during aging, Neurosci. Lett., 104 (1989) 309-313. 19 Price, M.T.C. and Cooper, R.M., U-shaped functions in a shockescape task, J. Cornp. Physiol. Psychol., 89 (1975) 600-606. 20 Randt, C.T., Barnett, B.M., Mc Ewen, B.S. and Quatermain, D., Amnesic effects of cycloheximide on two strains of mice with different memory characteristics, Exp. Neurol., 30 (1971) 467474. 21 Schwegler, H., Crusio, W.E. and Brust, I., Hippocampal mossy fibers and radial-maze learning in the mouse: a correlation with spatial working memory but not with non-spatial reference memory, Neuroscience, 34 (1990) 293-298. 22 Stanes, M.D., Brown, C.P. and Singer, G., Effect of physostigmine on Y-maze discrimination retention in the rat, Psychopharmacologia, 46 (1976) 269-276. 23 Staubli, U., Thibault, O., Di Lorenzo, M. and Lynch, G., Antagonism of NMDA receptors impairs acquisition but not retention of olfactory memory, Behav. Neurosci., 103 (1989) 54-60. 24 Teyler, T.J. and Discenna, P., Long-term potentiation as a candidate mnemonic device, Brain Res. Rev., 7 (1984) 15-28. 25 Ungerer, A., Mathis, C., M~lan, C. and De Barry, J., ROle des acides aminOsneuroexcitateurs dans les phOnom~nesde mOmoire, L'Enc~phale, 16 (1990) 423-429. 26 Ungerer, A., Mathis, C., MOlan, C. and De Barry, J., The NMDA receptor antagonists, CPP and 7-L-glutamyl-L-aspartate, selectively block post-training improvement of performance in a Y-maze avoidance learning task, Brain Res., 549 (1991) 59-65. 27 Ungerer, A., Schmitz-Bourgeois, M., MOlan, C., Boulanger, Y., Reinbolt, J., Amiri, I. and De Barry, J., 7-L-glutamyl-L-aspartate induces specific deficits in long-term memory and inhibits [3H]glutamate binding on hippocampal membranes, Brain Res., 446 (1988) 205-211.
69
BBR 01438
Strain-dependent effects of y-L-glutamyl-L-aspartate, a N M D A antagonist, on retention of a Y-maze avoidance learning task in mice A r i e l l e U n g e r e r '~, C l a u d i n e M r l a n a a n d J e a n D e B a r r y b a Laboratoire de Psychophysiologie, Universit~ Louis Pasteur, URA 1295 CNRS, Strasbourg (France) and b Centre de Neurochimie, CNRS, Strasbourg (France)
(Received 7 April 1992) (Revised version received 10 December 1992) (Accepted 4 February 1993) Key words: 7-L-Glutamyl-L-aspartate; Memory; Active avoidance; Inbred strain; N-Methyl-D-aspartate receptor; Mouse
The NMDA receptor antagonist, 7-L-glutamyl-L-aspartate (7-LGLA), suppressed spontaneous improvement in posttraining performance in Swiss mice during the hours following acquisition of a Y-maze avoidance learning task. Since variability in posttraining performance is at least partially due to genetic factors, we compared the effects of 7-LGLA on retention of Y-maze learning in C57BL/6J, DBA/2J and BALB/c mice. Mice had to leave the start alley of the maze within the first 5 s (temporal component) and to choose the left alley (spatial component). C57BL mice significantly improved their performance from 1 h to 24 h posttraining, whereas DBA/2J and BALB/c mice did not. However, only retention of the temporal component improved over time in C57BL. 7-LGLA administered immediately posttraining (0.025-25 #mol/kg, i.p.) dose-dependently impaired retention of the temporal component in C57BL mice 48 h later, but had no significant effect on retention of the spatial component. ~,-LGLA administered 24 h posttraining induced a similar but weaker deficit. In contrast, ~,-LGLA did not significantly affect retention of DBA/2J and BALB/c mice, regardless of the component analyzed or the time of administration. It had no effect on locomotor activity or emotional reactivity of animals of any strain. These results support the hypothesis of a specific action of y-LGLA on mechanisms involved in the treatment of information during the hours following acquisition, and suggest that NMDA receptors are involved in this action.
INTRODUCTION
Convergent data demonstrate that N-methyl-Daspartate ( N M D A ) receptors are critically involved in the induction of long-term potentiation (LTP) of synaptic responses at the hippocampal and cortical levels 1'3'9, suggesting that they may have a central role in learning and memory processes 13'24. This hypothesis originally received support from studies by Morris et al. ~7 who showed that the specific and competitive N M D A receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5) chronically infused in the lateral ventricle (ICV) blocked the induction of LTP and strongly disrupted acquisition of a spatial learning task in a water maze; D-AP5 did not affect acquisition per se, short-term memory or retrieval processes, but appeared to interfere specifically with formation of memory traces, which agrees with the hypothesis. In contrast, D-AP5 administered in the same conditions had no Correspondence: A. Ungerer, Laboratoire de Psychophysiologie, Universit6 Louis Pasteur, URA 1295 CNRS, 7 rue de l'Universit+, 67000-Strasbourg, France. Fax: (33) 88 35 84 49.
effect on acquisition of a visual discrimination in the water maze, suggesting a highly task-specific action 17. Task- and time-dependent effects of N M D A antagonists were subsequently confirmed in various learning tasks. For example, ICV infusion of D-AP5 retarded acquisition of an olfactory discrimination task, but had no effect on acquisition or retention of a one-way active avoidance learning z3. Elsewhere, pretraining administration of CGS 19755 (cis-4-phosphonomethyl-2piperidine carboxylate), a potent competitive N M D A antagonist, induced a marked retention deficit in a stepthrough passive avoidance task, but did not affect retention of a step-down passive avoidance learning ~2. Moreover, the noncompetitive N M D A antagonist, MK-801 [( + )-5-methyl- 10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5,10-imine maleate], impaired retention of a step-through dark avoidance learning task when administered prior to training and improved it when administered immediately posttraining, whereas both pre- and posttraining administration of MK-801 improved retention of a step-down passive avoidance task tr. Previous work suggests that N M D A receptors might
70 be specifically involved in mechanisms underlying longterm consolidation of memory traces, as indicated by behavioral effects of N M D A antagonists in a Y-maze avoidance learning task in which animals had to leave the start alley of the maze within the first 5 s (temporal component) and to choose the left alley (spatial component) to avoid foot-shock. Indeed, we showed that systemic posttraining administration of 7-L-glutamyl-L-aspartate (7-LGLA), a pseudo-peptide which has the parmacological properties of a competitive N M D A antagonist 14"26, specifically suppressed the spontaneous improvement in performance observed in control animals during the hours following acquisition27; 7-LGLA significantly impaired retention of the temporal component of the task, which improved significantly over time, whereas it had no effect on retention of the spatial component, which did not improve over time; moreover, ),-LGLA did not affect spatial recognition memory in an alternation task in which control animals showed no posttraining improvement in performance 25. Similar behavioral effects were observed following systemic administration of CPP ([3(2(carboxypiperazin-4-yl)propyl- 1-phosphonate], a potent and specific competitive N M D A antagonist, or ICV administration of D-AP5, suggesting a specific action of N M D A antagonists on mechanisms underlying spontaneous improvement in posttraining performa n c e 15,26.
Variability in posttraining performance over time is known to be at least partially due to genetic factors. The purpose of the present study, in light of these data, was to compare the effects of 7-LGLA on retention of a Y-maze avoidance learning task in three strains of mice, C57BL/6J, BALB/c and DBA/2J, which differ in mnemonic capacities and in posttraining evolution of performance 2'4't°'2°. A control experiment was also carried out using an open-field test procedure to determine whether memory deficits induced by ? - L G L A might be due to an action on locomotor and/or emotional reactivity of the animals. MATERIALS AND M E T H O D S
Animals Male mice of C57BL/6J, BALB/c, and DBA/2J strains, 6 weeks old, were purchased from IFFA-Credo (69210-1'Arbresle, France) and served as subjects. After arriving at the laboratory, they were housed under standard conditions on a 12:12-h light-dark cycle (lights on at 7.00 a.m. and off at 7.00 p.m.) with food and water ad libitum. The experiment started when they were 2 months old and weighed 25-35 g . Behavioral testing was carried out during the light phase of the cycle.
Y-maze avoidance learning task Mice were trained in a transparent Plexiglas Y-maze with three alleys (each 13 x4.5 × 5.5 cm) set on the median lines of an equilateral triangle. At the end of each alley was a transparent Plexiglas mobile box ( 1 0 x 4 . 5 x 5.5 cm) with an opaque Plexiglas sliding door which allowed transport of the animal from the goal alley to the start alley without handling. At the start of each trial, the animal entered the start alley from the mobile box and underwent a double conditioning procedure in which it had to leave the start alley within the first 5 s (temporal component) and to choose the left alley of the maze (spatial component) in order to avoid footshock (35 V AC, 4.5 mA; duration 1 s every 5 s). The animal was held to be making an avoidance error when it failed to leave the start alley within 5 s, a discrimination error when it entered the fight alley at least once, and an incorrect trial when it made either an avoidance error, or a discrimination error, or both. The animal underwent one trial every minute until it reached a criterion of 7 correct out of 8 consecutive trials. It was submitted to a second learning session at various delays following the first session in order to test retention. The number of incorrect trials, avoidance errors and discrimination errors made during the first and the second sessions were recorded. Statistical analyses used a two-factor ANOVA. Significant differences among groups were analyzed by the Newman-Keuls procedure. The Kruskal-Wallis test was used for analyses of discrimination errors during retention testing as their distribution was not normal in some groups. In the first experiment, four groups of mice of each strain were submitted to a first learning session and, 1 h, 3 h, 6 h, or 24 h later, to a second learning session in order to test retention. Each subject was tested only once. In the second experiment, immediately after the learning session mice of each strain received an intraperitoneal (i.p.) administration of 0.025, 0.25, 2.5 or 25 #mol/kg ?-LGLA dissolved in 0.9°Jo NaC1 (25 ml/kg). Control mice received 25 ml/kg 0.9°o NaC1. Learning retention was tested 48 h following the pharmacological treatment. In the third experiment, 7-LGLA (0.25, 2.5, 25 #mol/kg, i.p.) or vehicle was administered 24 h after the learning session and retention testing was carried out 48 h later. In the three experiments each group consisted of 11-12 mice. Exploratory activity in an open,field Behavioral effects of 7-LGLA on exploratory activity of C57BL/6J, BALB/c and DBA/2J mice were analyzed in a square open-field (50 × 50 x 50 cm) divided into 25 equal squares and including a central platform (8 x 8 x 4 cm) and 4 small pillars (2 × 2 × 6 cm) each set
71 at 10 cm from the two adjacent walls of the apparatus. Mice received an administration of either ~,-LGLA (0.25 or 25/~mol/kg, i.p.) or 0.9% NaC1 and 30 min later were placed in the apparatus for 30 min. The number of square crossings, rearings, crossings of the central platform and fecal boluses were recorded in every block of 5 min. Each group of mice contained 16 subjects. Data were analyzed by a two-factor ANOVA.
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Y-maze avoidance learning task Strain differences in acquisition and retention. Performance expressed by the number of incorrect trials made by C57BL/6J, BALB/c and DBA/2J mice during the first learning session and during retention testing carried out 1 h, 3 h, 6 h, or 24 h posttraining are shown in Fig. 1. Mice of the three strains showed significant differences in acquisition of the task (F2a2o=21.85; P<0.001) during the first learning session: DBA/2J mice made significantly fewer incorrect trials than BALB/c and C57BL mice, which did not differ from each other (Newman-Keuls test: DBA/2J vs. BALB/c P<0.01; DBA/2J vs. C57BL, P<0.01; BALB/c vs. C57BL, P>0.05). Performance also differed between strains in terms of the number of avoidance errors (F2,120 18.68; P < 0.001 ) and discrimination errors (F (2,120) -- 9.65; P < 0.001) (Fig. 2): DBA/2J mice made fewer avoidance errors than C57BL and BALB/c mice (Newman-Keuls test, P < 0.01), but C57BL made more avoidance errors than BALB/c mice ( P < 0.05); in contrast, C57BL mice did not make significantly more discrimination errors than DBA/2J mice, unlike BALB/c =
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mice (Newman-Keuls test, BALB/c vs. DBA/2J, P<0.01; BALB/c vs. C57BL, P<0.05; DBA/2J vs. C57BL, P>0.05). No significant difference was observed during the learning session between the four groups of each strain (1 h, 3 h, 6 h or 24 h) either for the number of incorrect trials (F3,12o = 0.917; P > 0.05), or the number of avoidance (F3a20 = 0.96) or discrimination e r r o r s (/173,120 = 0.41). Retention performance, expressed by the number of incorrect trials during the second learning session (Fig. 1) differed markedly across strains (F2a20 = 13.18; P < 0.001) and according to the time of retention testing (F3a2o=3.97; p=0.009), but no significant strain x time interaction was observed (F6,12o= 1.93; P>0.05). Comparisons between strains showed that DBA/2J and BALB/c mice did not differ significantly at any posttraining time. In contrast, C57BL mice showed impaired performance compared to DBA/2J and BALB/c mice 1 h and 3 h posttraining ( N e w m a n Keuls test, P<0.01), but not 6 h or 24 h posttraining. Moreover, comparisons within each strain showed that
72 C57BL mice significantly improved their performance from the first to the 24th hour following acquisition (Newman-Keuls test: 1 h vs. 24 h, P<0.01; 3 h vs. 24 h, P<0.01; 6 h vs. 24 h, P<0.05), whereas DBA/2J and BALB/c mice did not exhibit a time-dependent improvement in posttraining performance. The same pattern of results was obtained for the number of avoidance errors, which differed both across strains (F2,12o = 27.90; P<0.001) and according to the testing time (F3,120 = 8.20; P<0.001) (Fig. 2A): C57BL mice made more avoidance errors than DBA/2J and BALB/c mice at 1 h, 3 h, and 6 h posttraining (Newman-Keuls test, at 1 h: P<0.01; at 3 h: C57BL vs. DBA/2J, P<0.01; C57BL vs. BALB/c, P<0.05; at 6 h: C57BL vs. DBA/2J, P<0.05; C57BL vs. BALB/c, P<0.01), but not 24 h posttraining, and they showed significant improvement in performance between the first and the 24th hour following training (Newman-Keuls test, P < 0.01). DBA/2J and BALB/c mice did not improve performance and did not differ from each other at any time. In contrast, no significant difference in the number of discrimination errors (Fig. 2B) was observed between groups (Kruskal-Wallis test, H = 8.18, P > 0.05), indicating that the three strains did not differ for this parameter whatever the time of retention testing.
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Fig. 3. Dose-dependent effects of 7-LGLA on retention of a Y-maze avoidance learning task in C57BL, DBA/2J and BALB/c mice, as expressed by the number of incorrect trials (mean +_S.E.M.) made during retention testing. The pharmacological treatment was administered immediately following the learning session and retention was tested 48 h later.
mice whatever the dose administered and the parameter considered. In contrast, it induced a dose'dependent {SSS] NaCI t-'/q 0.025 0.25 E;~ 2.50
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Effects of posttraining administration of 7-LGLA on retention.There was no difference in acquisition of the task among experimental groups within each strain during the first learning session prior to 7-LGLA administration, but a strong strain effect was found for the number of incorrect trials (F2,,62 = 9.47, P < 0.001), the number of avoidance errors (F= 18.38) and the number of discrimination errors (F=6.05, P<0.01), thus confirming the results of the first experiment. 7-LGLA administered immediately posttraining deeply impaired retention performance 48 h later, as expressed by the number of incorrect trials made by the animals during retention testing. This deficit was both strain and dose-dependent (strain effect: F2.162 = 8.25, P < 0.001; dose effect: F4,162 = 7.73, P<0.001; strain x dose interaction, F8,162 = 0.78, P>0.05) (Fig. 3), But 7-LGLA appeared to affect primarily retention of the temporal component of the task, as shown by the significant increase in the number of avoidance errors (strain effect: F2,16 z = 17.34, P<0,001; dose effect: F4,16 2 = 8.90, P<0,001; strain x dose interaction, Fsa62 = 1.15, P > 0.05) (Fig. 4A), whereas the number of discrimination errors did not differ between groups (KruskalWallis test, H = 10.31, P > 0.05) (Fig. 4B), indicating that retention of the spatial component was not impaired. However, comparisons of experimental groups within each strain showed that 7-LGLA had no significant effect on learning retention in DBA/2J or BALB/c
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73 retention deficit in C57BL mice treated at doses of 0.25 to 25 #mol/kg 7-LGLA, as reflected by a significant increase in the number of incorrect trials and of avoidance errors made by these animals compared to control mice (Newman-Keuls test, for the two parameters: 0.25 7-LGLA vs. NaCI, P<0.05; 2.5 and 25 7-LGLA vs. NaCI, P < 0.01) (Fig. 3 and 4A). But, even in C57BL, 7-LGLA had no effect on the number of discrimination errors whatever the dose administered (H=3.52, P > 0 . 0 5 ) (Fig. 4B). To test whether retention deficits observed in C57BL mice were due to motor side effects, mice of this strain were submitted to the Y-maze learning task in the same conditions as above, except that they had to leave the start alley of the maze within the first 10 s, instead of 5 s, at the start of each trial. As observed in the preceding experiment, C57BL mice treated at the dose of 2.5 #mol/kg 7-LGLA immediately posttraining made a greater number of incorrect trials (F,.2o = 4.40; P < 0.05) and of avoidance e r r o r s ( F t , 2 0 = 7.68; P<0.025) than control animals, but did not differ from them in the number of discrimination errors, thus indicating that retention deficits induced by 7-LGLA are unlikely related to an action on motor activity. 7-LGLA administered 24 h after the first learning session had little effect on learning retention 48 h after pharmacological treatment, as measured by the number of incorrect trials (dose effect, F3,m7 = 2.05, P > 0.05) or discrimination errors (Kruskal-Wallis test, H = 6.35, P > 0.05) by treated and control animals; but it induced a strain- and dose-dependent increase in the number of avoidance errors (strain effect, F2,x27 = 24.75, P < 0.001; dose effect, F3,m7 = 4.32, P = 0.006; strain × dose interaction, F6.127=1.06, P > 0 . 0 5 ) (Fig. 5), which was
mainly due to impaired performance in C57BL mice treated at the higher dose of 7-LGLA (Newman-Keuls test: 25 7-LGLA vs. NaC1, P < 0.05). The other experimental groups did not differ from each other, regardless of strain or dose administered.
Effects of 7-LGLA on exploratory activity Behavioral effects of 7-LGLA were analyzed in mice submitted to an open-field test in order to determine whether retention deficit induced by the drug could be due to an action on some behavioral components that might interfere with the animal's performance. Results showed that C57BL, BALB/c and DBA/2J mice exhibited strong strain differences in locomotor activity, as expressed by the number of squares crossed during the first 5 min (strain effect, F2,~3s = 20.05, P < 0.001) or the total duration of the test (strain effect, F 2 , 1 3 5 = 16.85, P<0.001), DBA/2J mice being significantly more active than BALB/c or C57BL mice (Newman-Keuls test, P < 0.01). But 7-LGLA, administered at doses of 0.25 or 25 #mol/kg, had no significant effect either during the 5 first min (dose effect, Fz,~3s = 0.334, P > 0.05; strain x dose interaction, F4,135 -- 1.01, P>0.05) or the total duration of the test (dose effect, F2,~35= 1.38, P>0.05; strain x dose interaction, Fa,t35= 0.75, P > 0.05) (Fig. 6). Likewise, 7-LGLA had no effect on the number of rearings, crossings of the central platform, or boluses, whatever the strain or the dose used. Moreover, 7-LGLA, when administered 48 h prior to open-field testing at the dose of 25 #mol/kg, did not affect performance of C57BL mice, as expressed by the number of squares crossed during the first 5 min of the test (treated group, M = 218.81 + 12.47; control group, M = 238.68 + 12.62) or the number of rearings (treated
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Fig. 6. Effects of 7-LGLA on locomotor activity of C57BL, DBA/2J and BALB/c mice in an open-field. Results are expressed by the number of squares crossed (mean + S.E.M.) during the 30 min of testing. 7-LGLA (0.25 or 25/~mol/kg, IP) was administered 30 min before the beginning of the test.
74 group, M = 26.31 + 9.10; control group, M = 23.18 + 8.33). Thus, retention deficits observed in this strain of mice do not appear to be due to non-specific proactive effects of 7-LGLA on performance.
DISCUSSION
Among the three strains of mice used in the present study, only C57BL mice displayed spontaneous improvement in posttraining performance over time, thus confirming that such an improvement is controlled by genetic factors as shown by others 2'4'1°'2°. BALB/c mice acquired the task at a rate similar to that of C57BL mice and slower than DBA/2J mice, as shown by the number of incorrect trials during the learning session, but they did not show posttraining improvement in performance, in contrast to C57BL mice, and did not differ in retention performance from DBA/2J mice whatever the time of testing. Moreover, mice of the three strains differed significantly in acquisition of both the temporal and the spatial components of the task, but did not show differences on retention of the spatial component, whereas retention of the temporal component significantly improved over time in C57BL mice but not in the two other strains. These results clearly show a dissociation between the two components of the task, as retention of the temporal component improved during the hours following acquisition in C57BL mice, in contrast to retention of the spatial component which did not, confirming observations using the same task with Swiss mice 25'26. Posttraining improvement in performance is currently thought to reflect an organization process of memory traces and would be related to low levels of training during original learning 5'7'8. In light of these data, it must be emphasized that C57BL mice made significantly more avoidance errors than BALB/c and DBA/2J mice and more avoidance errors than discrimination errors during the learning session (F1.8o = 26.37, P < 0.001). This suggests that the temporal component is more complex than the spatial one and is acquired less in C57BL mice than in DBA/2J and BALB/c mice, which would explain why retention of this component improves over time in C57BL mice. In this way, the lack of posttraining improvement in performance of DBA/2J and BALB/c mice would be due to an overlearning of the two components of the task and not to an incapacity of these strains to exhibit such a phenomenon. In agreement with this hypothesis, DBA/2J and BALB/c mice have been shown to display timedependent improvement in posttraining performance in a two-way avoidance learning task 2 and in a lever-press
conditioning task 4, respectively. Moreover, C57BL mice did not show such an improvement in this latter t a s k 4 o r in a visual discrimination learning task ~~. These data clearly demonstrate that posttraining improvement in performance is highly dependent on experimental conditions, in addition to genetic factors. 7-LGLA, when administered immediately posttraining, deeply impaired retention of the temporal component in C57BL mice. But it did not affect retention of the spatial component in C57BL mice and had no significant effect on retention of DBA/2J and BALB/c mice whatever the learning component considered. Also, 7-LGLA had no effect on locomotor activity or emotional reactivity of any of the animals in open-field testing, thus confirming its lack of toxicity at the doses used and indicating that retention impairment observed in C57BL mice is not due to an action on these behavioral components. In fact, the retention deficit induced by 7-LGLA appears highly correlated with posttraining improvement in performance displayed by C57BL mice on retention of the temporal component. Moreover, 7-LGLA, at doses of 2.5 or 25 #mol/kg, induced a deficit in retention of the temporal component which had the same amplitude as that observed in C57BL mice tested 1 h posttraining: thus, 3,-LGEA completely suppressed improvement in performance displayed by control mice between the first and the 24th hour following the learning session. However, ?,-LGLA still induced a weak deficit in retention of the temporal component when administered 24 h posttraining in C57BL mice, suggesting that organization of memory traces is not completely achieved by then. Indeed, retention performance of the temporal component slightly improved in C57BL control mice between 24 and 48 h posttraining, which agrees with the hypothesis. Moreover, delays for maximum improvement in performance have been shown to vary according to the learning task and the degree of acquisition and to frequently exceed 24 h (refs. 5, 6, 19, 22). The selective action of 7-LGLA on retention of the temporal component in C57BL mice is similar to that induced by ,/-LGLA, CPP or D-AP5 in the same task, but in Swiss mice 15"26. These data thus strengthen the hypothesis that N M D A receptor antagonists specifically interfere with mechanisms underlying posttraining improvement in performance and that N M D A receptors are involved in this action. However, the question is raised whether interstrain differences in ?,-LGLA effects reflect genetic differences in excitatory amino acid systems, and more specifically in N M D A receptors. Unfortunately, we lack relevant data. A preliminary study carried out by us on hippocampal membranes showed that 7-LGLA displaced [3HI-L-glutamate in
75 the three strains, but no significant difference was observed in site densities or dissociation constant between them. Elsewhere, Peterson et al. ~8 did not find any differences in density or affinity of N M D A receptors between C57BL and BALB/c adult mice on a whole brain membrane preparation. However, it cannot be excluded that such differences may exist, for example in some subfields of the hippocampal formation. Indeed, recent work 2~ showed that the size of the mossy fiber terminal fields varies across strains of mice, including C57BL, BALB/c and DBA/2J and is negatively correlated with performance in a spatial working memory task. In light of these data, interstrain studies on N M D A receptor pharmacology may be a valuable tool for a better understanding of these receptors and of their role in learning and memory processes.
ACKNOWLEDGEMENTS
This work was supported by D R E T (85/158, 88/ 057). We thank Dr. A. Mann for synthetizing ~-LGLA and Dr. J. Anderson for his help in carefully reading the manuscript.
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