Involvement of hippocampal NMDA receptors in retrieval of spontaneous object recognition memory in rats
Etsushi Iwamura Kazuo Yamada Yukio Ichitani
Highlights
• Hippocampal NMDA receptors were blocked during test phase of spontaneous object recognition.
• Drug effects depended on delay length and number of sample exposures.
• Hippocampal NMDA receptors are important for retrieval of long-term object memory.
Abstract
The involvement of hippocampal N-methyl-D-aspartate (NMDA) receptors in the retrieval process of spontaneous object recognition memory was investigated. The spontaneous object recognition test consisted of three phases. In the sample phase, rats were exposed to two identical objects several (2-5) times in the arena. After the sample phase, various lengths of delay intervals (24 hours-6 weeks) were inserted (delay phase). In the test phase in which both the familiar and the novel objects were placed in the arena, rats’ novel object exploration behavior under the hippocampal treatment of NMDA receptor antagonist, AP5, or vehicle was observed. With 5 exposure sessions in the sample phase (experiment 1), AP5 treatment in the test phase significantly decreased discrimination ratio when the delay was 3 weeks but not when it was one week. On the other hand, with 2 exposure sessions in the sample phase (experiment 2) in which even vehicle-injected control animals could not discriminate the novel object from the familiar one with a 3 week delay, AP5 treatment significantly decreased discrimination ratio when the delay was one week, but not when it was 24 hours. Additional experiment (experiment 3) showed that the hippocampal treatment of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, NBQX, decreased discrimination ratio with all delay intervals tested (24 hours-3 weeks).
Results suggest that hippocampal NMDA receptors play an important role in the retrieval of spontaneous object recognition memory especially when the memory trace weakens.
Keywords: spontaneous object recognition memory, retrieval, hippocampus, NMDA receptor, AMPA receptor, rats.
1. Introduction
It has been suggested that N-methyl-D-aspartate (NMDA) receptors, a kind of glutamate receptors, play an important role in long-term potentiation (LTP)[1], which is regarded as a neural base of learning and memory. Many previous studies have shown that hippocampal or intraventricular treatment of NMDA receptor antagonists impairs acquisition of or performance in learning and memory tasks such as radial maze and Morris water maze tasks, suggesting that hippocampal NMDA receptors paly an important role in learning and memory especially in spatial memory [1-4]. However, what stage(s) of memory, i.e. encoding, consolidation, retention and retrieval, NMDA receptors are mainly involved in remains debated [5-8].
The involvement of hippocampal NMDA receptors in the retrieval process of memory is not so clear as in the encoding and consolidation processes. A previous study using CA3-NR1 knockout mice [9] showed evidence that CA3 NMDA receptors are involved in the retrieval process of spatial memory in Morris water maze. Fellini et al. [7], using an injection of DL-2-amino-5-phosphonopentanoic acid (AP5), a NMDA receptor antagonist, into CA3 of the dorsal hippocampus (HPC) suggested that CA3 NMDA receptors are crucial during long-term memory retrieval in Morris water maze task particularly when the amount of environmental information available is strongly limited. Furthermore, in a delay-interposed radial maze task, in which rats needed to retrieve their own choice of the first-half performance to efficiently get reward in the second-half performance, hippocampal AP5 treatment immediately before the second-half performance lowered correct choice responses [8] suggesting that hippocampal NMDA receptor activation is necessary for the retrieval of the first-half performance. These previous studies suggest the possibility that NMDA receptors are involved in the retrieval process of memory. However, since all these studies employed spatial tasks, expending this conclusion lead from these studies into non-spatial memory is still in debate.
Using the spontaneous object recognition (SOR) test is one strategy to address this issue. In this test, animals’ exploratory behavior to discriminate between the familiar and the novel objects is an index of memory [10], and they do no need to process spatial information. Involvement of HPC in performance of the SOR is still controversial [11-18]. However, the role of HPC in SOR memory has been suggested especially when the retention interval is long enough [17,18]. Cohen et al. [19] used a intrahippocampal muscimol microinfusion to transiently inactivate the mouse HPC at distinct stages during the SOR test, and showed that the HPC is important not only for the encoding and consolidation but also for the retrieval of the SOR memory. However, the role of hippocampal NMDA receptors in the retrieval of SOR memory is still unclear. Additionally, the retention interval tested in their study was at most 24 hours (hr).
In the present study, therefore, we addressed whether NMDA receptors in the HPC are essential for the retrieval process of SOR memory using a hippocampal microinfusion of an NMDA receptor antagonist AP5 with longer retention intervals, since there is a possibility that the involvement of hippocampal NMDA receptors in the retrieval process is dependent on the length of retention interval. However, if the effect of NMDA receptor blockade on SOR performance is determined by the strength of memory trace at the retrieval time, repetitions of encoding in the sample phase are also supposed to affect. Thus, we conducted two experiments based on the number of encoding sessions (experiments 1 and 2). Additionally, we also investigated the role of hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the SOR memory retrieval using an AMPA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) disodium (experiment 3).
2. Materials and Methods
2.1. Animals
Male Wistar-Imamichi rats (n = 82) were used as subjects. They were housed individually and kept on a 12 hr light-dark cycle with continuous access to food and water. All behavioral testing was conducted during the light phase (08:00-20:00). Animal experiments were approved by the University of Tsukuba Committee on Animal Research. All efforts were made to minimize the number of animals used and their suffering.
2.2. Surgery and histology
Under sodium pentobarbital anesthesia (40 mg/kg, i.p.) using a stereotaxic instrument (David Kopf, CA), guide cannulae were bilaterally implanted into the dorsal HPC (AP -4.3 mm, LM ±2.7 mm from bregma, DV -3.2 mm from skull surface, skull flat), and fixed by dental cement and four small screws. Postoperative recovery period was 7 days. After the completion of behavioral tests, rats were deeply anesthetized with sodium pentobarbital, and perfused intracardially with 0.9% saline solution followed by 10% formalin solution. The brains were removed and postfixed in formalin solution for 24 hr and then immersed in 20% sucrose solution. Coronal sections (40 µm) were cut in a cryostat (CM3000, Leica, Wetzlar), and stained with cresyl violet. Sections were examined under a light microscope to access the location of tips of injection cannulae.
2.3. Drugs
AP5 (20 mM; Sigma, MO) and NBQX (2.5 mM; Sigma, MO) were dissolved in 0.02 M phosphate buffer (PB). Rats received bilateral intrahippocampal infusion of a drug (1.0 µl/side, 0.5 µl/min) or vehicle (0.02 M PB) using a microsyringe pump (EP-50, EICOM, Kyoto) 15 min before the test phase. For intrahippocampal infusion, injection cannulae were inserted into the guide cannulae and advanced 1.0 mm below the tips of them. After the drug injection, the injection cannulae were kept in place for additional one min to allow the diffusion of the drug. A previous autoradiography study investigated in detail the spread of intrahippocampal [³H] D-AP5 [6], and showed the diffusion of the similar amount of drug was restricted to the dorsal HPC.
2.4. Spontaneous Object Recognition (SOR) Test
2.4.1. Apparatus
An open-field arena (90 × 90 × 45 cm) made of polyvinylchloride was used. The walls of the arena were colored in black, while the floor was gray. An overhead camera and a video recorder were used to monitor and record the animals’ behavior for the analysis. The illumination of the center of the arena was 50 lx. The stimulus objects were copies of 10 different objects made of glass, metal, or plastic and varied in height between 7–15 cm. All the objects were heavy enough or fixed on the heavy metal plate so that the rats could not move them during testing.
2.4.2. General procedure
Prior to the SOR test, rats received 5 min of handling and were habituated to the arena without any objects for 10 min for 3 consecutive days. The SOR test consisted of a sample phase (2-5 sample exposure sessions) and a test phase, and these phases were separated by a delay interval. In the sample phase, two identical objects were placed diagonally in the arena (the center of each object was 22.5 cm from adjacent two walls), and the rat was released in the center of the arena. Each rat was allowed to explore freely these objects for 5 min. After the sample phase, the rat was removed and returned to its home cage. In the test phase, two objects were placed at the same positions as in the sample phase. One object was the familiar object, which was presented in the sample phase, and the other was a novel object. The positions of the novel objects in the test phase were counterbalanced. The animal was placed into the arena and allowed to explore the objects for 5 min.
In both the sample and test phases, we measured the time rats spent exploring the objects. Exploration was defined as the rat directing its nose toward the object within a distance of 2 cm. Touching the object with other parts of the body or climbing over the object was not included. In the test phase, the discrimination ratio (DR) was calculated by dividing the amount of exploration of the novel object by the total amount of exploration of two objects (the familiar and the novel objects). The analysis was conducted on the first 2 min of the test phase, during which rats’ preference for the novel object is typically greatest [20, 21]. The arena was wiped with a wet cloth containing sodium hypochlorite solution and the objects were cleaned up with 70% ethanol to eliminate odor cues.
2.4.3. Experiment 1: Effects of AP5 on SOR with 5 sample exposure sessions (Fig. 1A)
In the sample phase, rats explored a pair of identical objects (A) in the arena for 5 min repeatedly for 5 times. The intersession interval was approximately 30 min. On the next day, another pair of objects (B) was used as the sample stimuli. After a pre-defined delay interval, the test phase was conducted once a day for 2 consecutive days. One of the sample objects was tested on the first day (A vs C), and the other was tested on the next day (B vs D). AP5 or PB was infused 15 min prior to each test phase. The order of the sample objects (A or B) and the drug treatments (AP5 or PB) was counterbalanced. That is, half of the animals were injected with AP5 and the other half were injected with PB on the first test day (Test 1). In each drug condition, half of the animals were tested with object A, and the other half were tested with B. In the second test day (Test 2), the rest of drug conditions (PB/AP5) with the rest of objects (B/A) was tested. The animals were randomly assigned to 1 week (wk)- (n = 15), 3 wk- (n = 13) or 6 wk-delay (n = 11) group. Intrahippocampal cannulation was conducted 1 wk before the test phase (3 wk- and 6 wk-delay groups), or 1 wk before the sample phase (1 wk-delay group).
2.4.4. Experiment 2: Effect of AP5 on SOR with 2 sample exposure sessions
Fourteen rats were assigned to the 1 wk- (n = 6) or 3 wk-delay (n = 8) group. The procedure was the same as in experiment 1, but rats explored a pair of identical objects for 5 min twice (Fig. 1A). AP5 or PB was infused 15 min prior to each test phase. Rats of the 3 wk-delay group were then given two 24 hr-delay tests using different object pairs (Fig. 1B). One test was for AP5 treatment, and the other was for PB treatment. Objects were presented twice in the sample phase in the same manner as in the 3 wk-delay test.
2.4.5. Experiment 3: Effects of NBQX on SOR with 5 sample exposure sessions
Rats were randomly assigned to the 24 hr- (n = 10), 1 wk- (n = 10) or 3 wk-delay (n = 9) group to test the effect of NBQX. The procedure was the same as in experiment 1 except that NBQX or PB was infused 15 min prior to each test phase and additional 24 hr-delay tests were not conducted. Since experiment 1 showed that rats in the 6 wk-delay group could not discriminate a novel object from the familiar one, we did not make the 6 wk-delay group in this experiment.
2.5. Statistical analysis
Exploration time in the SOR test was analyzed using a two-way analysis of variance (ANOVA) (Object × Drug) followed by a post-hoc Bonferroni test. DRs were also analyzed using a two-way ANOVA (Drug × Delay) followed by a post-hoc Bonferroni test. In the SOR test, it was suggested more appropriate to evaluate rats’ recognition memory based on whether a group’s average DR significantly differs from chance or not, rather than comparing DRs among conditions [22]. Therefore, DRs were also compared to the theoretical chance level (50%) using a one-sample t-test.
3. Results
3.1. Histology
The locations of tips of injection cannulae are shown in Fig. 2. All tips in experiments 1-3 were identified in the dorsal HPC.
3.2. Experiment 1
Exploration time in the 5 sample sessions across 2 days is shown in Fig. 3. In each day, exploration time in the first session was longest and it gradually decreased in the following sessions. According to a three-way ANOVA (Delay group × Day × Session), neither main effects of Delay nor Day were significant. Only the main effect of Session was significant [F (4, 144) = 36.43, p < .01]. This suggests that exploration time significantly decreased across 5 sessions in each day, but exploration time for the first and second objects (Day 1 and Day 2 respectively) were not different. Additionally, it was shown that exploration time of objects in the sample phase was not different among three delay groups.
Since a previous study revealed that the measure using exploration time itself was vulnerable to differences in exploration levels, in the next analysis we calculated DR scores, which correct for exploratory differences [23]. The mean DRs of three interval groups in the test phase are shown in Fig. 4B. A two-way ANOVA showed a significant main effect of Delay [F (2, 36) = 4.53, p < .05] and a significant interaction of Drug × Delay effects [F (2, 36) = 7.08, p < .01]. Further analysis of simple main effects revealed that DR of AP5 condition in the 3 wk-delay group was significantly lower than that of PB condition (p < .01). In comparison with chance level of DR (50%), DRs of both PB and AP5 conditions in the 1 wk-delay group as well as PB condition in the 3 wk-delay group were significantly higher than chance (t-test, p < .01). DRs in other conditions were not significantly different from chance level.
3.3. Experiment 2
The mean DRs in the test phase are shown in Fig. 5B. A two-way ANOVA using data in the 1 wk- and 3 wk-delay groups showed a significant main effect of Drug [F (1, 12) = 10.48, p < .01], but the effect of Delay and the interaction between two factors were not significant. In comparison with chance level, DRs in both conditions of the 24 hr-delay test (PB: p < .01, AP5: p < .05) and the PB condition of the 1 wk-delay group (p < .05) were significantly higher than chance. DRs in all other conditions were not significantly different from chance level.
3.4. Experiment 3
The mean DRs in the test phase are shown in Fig. 6B. A two-way ANOVA showed significant main effects of Drug [F (1, 26) = 15.08, p < .01] and Delay [F (2, 26) = 3.59, p < .05]. No significant interaction between the two factors was detected. In comparison with chance level, DRs of PB condition in all delay groups were significantly higher than chance level (24 hr: p < .01, 1 wk: p < .05, 3 wk: p < .01). However, all DRs under NBQX condition were not different from chance level.
4. Discussion
In the present study, when objects were presented repeatedly 5 times in the sample phase, rats showed novel object preference in the test phase after a 1-3 wk delay, but not after a 6 wk delay, under PB treatment (Fig. 4B). On the other hand, when objects were presented only twice in the sample phase, rats showed novel object preference after a 24 hr-1 wk delay, but not after a 3 wk delay (Fig. 5B). These results show that SOR memory could be retained longer than that tested in most previous studies, depending on the number of exposure (encoding) sessions in the sample phase. Some previous studies also reported that the rat’s SOR memory was retained for 7-10 wk after the sample phase [17,18]. The discrepancy between the possible retention interval of 3 wk in the current study and the longer retention intervals in the previous reports may come from a difference of procedure in the sample phase: rats in our study explored objects 5 times repeatedly within a day, while rats in the previous studies explored sample object 5 min a day for 5 consecutive days.
We investigated the role of HPC in the SOR using AMPA receptor blockade (experiment 3). Rats were tested with 24 hr-3 wk delay intervals, and all groups showed a good novel object preference under PB treatment. NBQX impaired the SOR memory in all delay conditions (Fig. 6B) suggesting that the HPC is important for the retrieval process of SOR memory under a wide range of retention intervals. A previous study using hippocampal muscimol infusions confirmed that the HPC is important for the retrieval of SOR memory [19]. Our present result is consistent with the previous study and further revealed that involvement of the HPC in the SOR memory retrieval extends to at least 3 wk.
In the current study, hippocampal AP5 treatment before the test phase decreased novel object preference (DR score) suggesting that hippocampal NMDA receptors are involved in the retrieval process of SOR memory. However, the effect of AP5 depended on both the length of delay and the number of encoding sessions in the sample phase. When rats were exposed to objects 5 times in the sample phase, AP5 during the test phase did not decrease DR after a 1 wk delay, but did decrease it after a 3 wk delay (Fig. 4B). On the other hand, when rats were exposed to objects only twice in the sample phase, AP5 decreased DR to the level that was not different from chance after a 1 wk delay, but not after a 24 hr delay (Fig. 5B). In other words, the less the animals explored sample objects, the shorter delay interval AP5 treatment impaired the retrieval of SOR memory with. In general, it is considered that multiple object presentations could extend memory retention and longer delay intervals would decrease the memory trace. Thus, it is plausible that AP5 impaired the memory retrieval when the memory trace was getting weaker. That is to say, the dorsal hippocampal NMDA receptors may play an important role in the retrieval of SOR memory that has decayed under a threshold level. It is well known that NMDA receptors are activated following sufficient activation of AMPA receptors, and LTP can be elicited by the activated NMDA receptors. Most of research on the NMDA receptor involvement in LTP or memory function have focused on encoding and/or consolidation processes. With respect to studies on the SOR, systemic treatment of noncompetitive NMDA receptor antagonist MK-801 impaired both short- and long-term retentions of the SOR in rats when given either before or after the sample phase [24]. This suggests that NMDA receptor activation is necessary for formation of the SOR memory. Focusing on the role of hippocampal NMDA receptors, CA1-specific NMDA receptor 1 (NR1) subunit-knockout mice showed impaired SOR [25]. Additionally, using an intrahippocampal injection of AP5, Baker and Kim [26] suggested that the hippocampal NMDA receptors are important for encoding and consolidation of the SOR memory. However, the role of hippocampal NMDA receptors in the retrieval process has not been as clear as in the encoding or consolidation process. Evidence that the CA3 NMDA receptors are involved in the retrieval process of spatial memory in Morris water maze has been obtained using CA3-NR1 knockout mice [9] or AP5 treatment into CA3 of the dorsal HPC [7]. Our present results using the SOR memory situation showed, for the first time, evidence that non-spatial memory retrieval also requires the hippocampal NMDA receptor activation when the memory trace is probably getting weaker after a long retention interval.
One possible explanation why AP5 did not impair memory retrieval after relatively a short delay interval in the present study comes from evidence of the role of AMPA and NMDA receptors at the postsynaptic density (PSD). Lopez et al. [27] showed that trafficking of AMPA receptors to the PSD was essential for fear memory retrieval, and this trafficking was mediated by activated NMDA receptors. This suggests that NMDA receptor activation is necessary for the retrieval process. Additionally, it also predicts that NMDA receptor blockade will be ineffective when the level of AMPA receptors at the PSD is high enough. Retrieval of the SOR memory after a short delay interval, in which drug-induced NMDA receptor blockade did not have any substantial effect in the present study, might be an example of this situation. However, whether the level of AMPA receptors at the PSD gradually decreases along with time passage after memorization remains to be clarified before this interpretation can be accepted.
References
[1] Morris RGM, Anderson E, Lynch GS, Baudry M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 1986;319:774-6.
[2] Kawabe K, Ichitani Y, Iwasaki T. Effects of intrahippocampal AP5 treatment on radial-arm maze performance in rats. Brain Res 1998;781:300-6.
[3] Morris RGM. Synaptic plasticity and learning: selective impairment of learning in rats and blockade of long-term potentiation in vivo by the N-methyl-D-aspartate receptor antagonist, AP5. J Neurosci 1989;9:3040-57.
[4] Morris RGM, Halliwell RF, Bowery N. Synaptic plasticity and learning Ⅱ: Do different kinds of plasticity underlie different kinds of learning? Neurophychologia 1989;27:41-59.
[5] Izquierdo LA, Barros DM, Ardenghi PG, Pereira P, Rodrigues C, Choi H, Medina JH, Izquierdo I. Different hippocampal molecular requirements for short- and long-term retrieval of one-trial avoidance learning. Behav Brain Res 2000;111:93-98.
[6] Steele RJ, Morris RGM. Delay-dependent impairment of a matching-to-place task with chronic and intrahippocampal infusion of the NMDA-antagonist D-AP5. Hippocampus 1999;9:118-36.
[7] Fellini L, Florian C, Courtey J, Roullet P. Pharmalogical intervention of hippocampal CA3 NMDA receptors impairs acquisition and long-term memory retrieval of spatial pattern completion task. Learn Mem 2009;16:387-94.
[8] Yoshihara T, Ichitani Y. Hippocampal N-methyl-D-aspartate receptor-mediated encoding and retrieval processes in spatial working memory: delay-interposed radial maze performance in rats. Neuroscience 2004;129:1-10.
[9] Nakazawa K, Quirk MC, Chitwood RA, Watanane M, Yeckel MF, Sun LD, Kato A, Carr CA, Johnston D, Wilson MA, Tonegawa S. Requirement for hippocampal CA3 NMDA receptors in associative memory recall. Science 2002;297:211-8.
[10] Ennaceur A, Delacour J. A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav Brain Res 1988;31:47-59.
[11] Winters BD, Forwood SE, Cowell RA, Saksida LM, Bussey TJ. Double NBQX dissociation between the effects of peri–postrhinal cortex and hippocampal lesions on tests of object recognition and spatial memory: heterogeneity of function within the temporal lobe. J Neurosci 2004;24:5901-8.
[12] Forwood SE, Winters BD, Bussey TJ. Hippocampal lesions that abolish spatial maze performance spare object recognition memory at delays of up to 48 hours. Hippocampus 2005;15:347-55.
[13] Mumby DG, Tremblay A, Lecluse V, Lehmann H. Hippocampal damage and anterograde object-recognition in rats after long retention intervals. Hippocampus 2005;15:1050-6.
[14] Clark RE, Zola SM, Squire LR. Impaired recognition memory in rats after damage to the hippocampus. J Neurosci 2000;20:8853-60.
[15] Broadbent NJ, Squire LR, Clark RE. Spatial memory, recognition memory, and the hippocampus. Proc Natl Acad Sci USA 2004;101:14515-20.
[16] Barker GRI, Warburton EC. When is the hippocampus involved in recognition memory? J Neurosci 2011;31:10721-31.
[17] Gaskin S, Tremblay A, Mumby DG. Retrograde and anterograde object recognition in rats with hippocampal lesions. Hippocampus 2003;13:962-9.
[18] Broadbent NJ, Gaskin S, Squire LR, Clark RE. Object recognition memory and the rodent hippocampus. Learn Mem 2010;17:5-11.
[19] Cohen SJ, Munchow AH, Rios LM, Zhang G, Asgeirsdóttir HN, Stackman RW Jr. The rodent hippocampus is essential for nonspatial object memory. Curr Biol 2013;17:1685-90.
[20] Dix SL, Aggleton JP. Extending the spontaneous preference test of recognition: evidence of object-location and object-context recognition. Behav Brain Res 1999;99:191-200.
[21] Mumby DG, Gaskin S, Glenn MJ, Schramek TE, Lehmann H. Hippocampal damage and exploratory preferences in rats: memory for objects, places, and contexts. Learn Mem 2002;9:49-57.
[22] Gaskin S, Tardif M, Cole E, Piterkin P, Kayello L, Mumby DG. Object familiarization and novel-object preference in rats. Behav Processes 2010;83:61-71.
[23] Akkerman S, Prickaerts J, Steinbusch HWM, Blokland A. Object recognition testing: Statistical considerations. Behav Brain Res 2012;232;317-22.
[24] de Lima MN, Laranja DC, Bromberg E, Roesler R, Schröder N. Pre- or post-training administration of the NMDA receptor blocker MK-801 impairs object recognition memory in rats. Behav Brain Res 2005;156:139-43.
[25] Rampon C, Tang YP, Goodhouse J, Shimizu E, Kyin M, Tsien Jr. Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice. Nat Neurosci 2000;3:238-44.
[26] Baker KB, Kim JJ. Effects of stress and hippocampal NMDA receptor antagonism on recognition memory in rats. Learn Mem 2002;9:58-65.
[27] Lopez J, Gamache K, Schneider R, Nader K. Memory retrieval requires ongoing protein synthesis and NMDA receptor activity-mediated AMPA receptor trafficking. J Neurosci 2015;35:2465-75.
[28] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 4th ed. San Diego: Academic Press. 1998.