Behavioural Brain Research 259 (2014) 261–267
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Research report
Post-acquisition hippocampal NMDA receptor blockade sustains retention of spatial reference memory in Morris water maze Keisuke Shinohara ∗ , Toshimichi Hata Faculty of Psychology, Doshisha University, Kyotanabe, Japan
h i g h l i g h t s • Post-acquisition hippocampal NMDAR blockade suppresses decay of spatial memory. • The memory, once it had deteriorated, was not enhanced by this treatment. • Hippocampal NMDARs are important for deterioration of spatial reference memory.
a r t i c l e
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Article history: Received 28 August 2013 Received in revised form 6 November 2013 Accepted 11 November 2013 Available online 17 November 2013 Keywords: Forgetting Spatial reference memory NMDA receptor Hippocampus
a b s t r a c t Several studies have demonstrated that the hippocampal N-methyl-d-aspartate type glutamate receptors (NMDARs) are necessary for the acquisition but not the retention of spatial reference memory. In contrast, a few studies have shown that post-acquisition repetitive intraperitoneal injections of an NMDAR antagonist facilitate the retention of spatial reference memory in a radial maze task. In the present study, we investigated the role of hippocampal NMDARs in the retention of spatial reference memories in Morris water maze. In Experiment 1, 24 h after training (4 trials/day for 4 days), d-AP5 was chronically infused into the hippocampus of rats for 5 days. In the subsequent probe test (seven days after training), we found that rats infused with d-AP5 spent a significantly longer time in the target quadrant compared to chance level, whereas rats in the control group did not. In Experiment 2, d-AP5 was infused into the hippocampus 1 (immediate) or 7 (delayed) days after the training session. In the probe test, following the retention interval of 13 days, immediate infusion facilitated the performance in a manner similar to Experiment 1, whereas the delayed infusion did not. These findings suggest that hippocampal NMDARs play an important role in the deterioration of spatial reference memory. © 2013 Elsevier B.V. All rights reserved.
1. Introduction A number of studies have demonstrated that the pharmacological blockade of N-methyl-d-aspartate type glutamate receptors (NMDARs) impairs the acquisition of spatial reference memory [1–8]. However, Villarreal et al. [9] demonstrate that the postacquisition administration of an NMDAR antagonist facilitated the retention of spatial reference memory. In their study, rats were trained in an 8-arm radial maze task (4 arms baited) and were then administrated repetitive intra-peritoneal injections of an NMDAR antagonist, (R,S)-3-(2-carboxypiperazin-4-yl) propyl1-phosphonic acid (CPP), during a 5-day retention period. In the subsequent retention test, fewer errors were committed by the CPP-treated rats than by the control rats. However, they did not
∗ Corresponding author at: Faculty of Psychology, Doshisha University, 1-3 TataraMiyakodani, Kyotanabe 610-0394, Japan. Tel.: +81 0774 65 7087; fax: +81 0774 65 7250. E-mail address: [email protected] (K. Shinohara). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.11.016
specify whether the effect was present in working or reference memory, because the performance in this task is dependent on both types of memory. To our knowledge, only few studies have supported the findings of the previously mentioned study [9], using tasks that are thought to primarily involve spatial reference memory. Namely, blocking NMDARs after the acquisition of spatial reference memory does not affect retention [10–13]. For example, in the Morris water maze place task, an NMDAR antagonist, d-2-amino-5-phosphonovaleric acid (d-AP5), was chronically infused into the lateral ventricle for 7 days following training. Treated rats performed similarly to controls on a retention probe test conducted 14 days after training [11]. This result seems to conflict with that of Villarreal et al. [9]. However, these studies cannot be compared, because there was at least one major methodological difference between them. The key variable may be the retention interval period. The retention interval period mentioned in Villarreal et al. [9] was such that the memories of the control group at the time of the retention test had already decayed back to the baseline level. However, other studies [10–13] reported that the memory was still
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above the baseline. In the latter cases, even if the post-acquisition NMDAR blockade had memory facilitation effects, it might have been masked by a ceiling effect. However, the study by Villarreal et al. [9] allows the detection of any possible memory facilitation effects of NMDAR antagonists. Therefore, the findings of Villarreal et al. [9] and other study findings [10–13] cannot be compared. In light of this unresolved issue in the literature, we previously used a Morris water maze to examine the effect of post-acquisition NMDAR blocking on retention test performance with respect to spatial memory, which persisted for 1 day but decayed within 7 days [14]. In such a situation, we demonstrated that chronic infusion of d-AP5 in the lateral ventricle post-acquisition maintained the memory for platform location in the probe test at seven days after training. This finding suggested that memory facilitation effects of the post-acquisition NMDAR blockade can be observed when the acquired memory has decayed in normal animals. Which brain structure functions as the critical site for the facilitating effect of NMDAR blockers? The hippocampus is a predominant candidate, since it has been closely involved in the acquisition of spatial reference memory [15,16]. However, the sites that are affected by NMDAR blocking have not yet been confirmed. This is because in studies that have reported memory facilitation effects, the NMDAR blockers were infused intraperitoneally [9] or intraventricularly [14]; therefore, affecting NMDARs throughout the whole brain. In the present study, we chronically infused d-AP5 into the dorsal hippocampus to elucidate the role of hippocampal NMDAR in the deterioration of spatial reference memory after its acquisition. We demonstrate, for the first time, that the chronic infusion of d-AP5 into the dorsal hippocampus after training suppresses the deterioration of spatial reference memory. These findings suggest that hippocampal NMDARs play an important role in the decay of spatial reference memory.
2. Materials and methods
2.2. Apparatus A circular pool, 140 cm in diameter with a 50-cm-high wall, was used. Both the pool floor and wall were black. The pool was filled with water (24 ± 1 ◦ C) to a depth of 13 cm and made opaque by black carbon ink. A number of environmental cues were present in the experimental room. The escape platform was a black cylinder 12 cm in diameter and 11 cm in height. It was submerged 2 cm below the water surface in the center of a specific quadrant. This shallower pool, compared with other water maze studies [17], can be used as a tool to assess rat behavior in the water maze because male adult Wistar rats can successfully remember the location of the platform at a depth of around 10 cm [18]. The shallow depth of the water maze has an advantage over a deep one in that the quality and temperature of the water can be managed more easily. 2.3. General behavioral procedures 2.3.1. Training session Following handling (5 min/day for 5 days), rats were trained in four trials (with at least a 10-min inter-trial interval) per day for 4 days. In the acquisition training trials, the location of the submerged escape platform was fixed at the center of a quadrant (target quadrant; TQ) for each rat. The rat was placed in the water facing the wall of the pool at one of the four starting points (north, east, south, or west). If the rat could not find the platform within 60 s, it was gently guided to the platform by the experimenter. The rat was left on the platform for 15 s and then removed from the pool and dried with a towel. 2.3.2. Probe test After a retention interval of 7 or 13 days, a probe test was conducted. In the probe test, the rats were allowed to swim freely without the platform for 60 s. The starting position was the point opposite the target. The time spent in TQ and the number of goal crossings (traversing the actual location of the escape platform) were recorded.
2.1. Subjects Fifty-eight naïve male albino Wistar rats (Shimizu, Kyoto, Japan) were used for this experiment; however, four rats were excluded from the analysis as their hippocampi were injured due to chronic infusion. At the beginning of training, all rats were 10–11 weeks old. Rats were housed individually with food and water available ad libitum and a controlled 12-h light–dark cycle (from 8:00 AM to 8:00 PM). Experiments were conducted during the light period (from 10:00 AM to 2:00 PM). All procedures and treatments were approved by the Doshisha Committee of Animal Experiment. In Experiment 1, 24 rats were divided into three groups, artificial cerebral spinal fluid (aCSF) infused (aCSF; n = 8), 15 mM d-AP5infused (n = 8), or 30 mM d-AP5-infused (n = 8), groups in such a way that the mean escape latencies in the last training day were counterbalanced. The average body weight (mean ± standard deviation [SD]) of the aCSF, 15 mM d-AP5, and 30 mM d-AP5 groups were 276.8 ± 14.9 g, 273.8 ± 15.8 g, and 274.9 ± 21.4 g, respectively. In Experiment 2, 30 rats were divided into an immediate aCSF-infused (Imm-aCSF; n = 7), an immediate 15 mM d-AP5-infused (Imm-AP5; n = 8), a delayed aCSF-infused (DelaCSF; n = 7), and a delayed 15 mM d-AP5-infused (Del-AP5; n = 8) group. The average body weight (mean ± SD) of the Imm-aCSF, Imm-AP5, Del-aCSF, and Del-AP5 groups were 273.4 ± 13.7 g, 265.5 ± 10.7 g, 273.4 ± 10.4 g, and 276.8 ± 11.5 g, respectively.
2.3.3. Drug preparation d-AP5 (Enzo Life Sciences, Inc., USA) was dissolved in aCSF and 15 mM and 30 mM solutions were prepared. Before implantation, minipumps were filled with the solution and incubated at 37 ◦ C for 24 h for priming and stabilization of the flow. 2.3.4. Surgery Rats received an intraperitoneal injection of atropine sulfate (0.1 mL; Tanabe Seiyaku, Japan), were anesthetized with sodium pentobarbital (60 mg/kg; Kyoritsu Seiyaku, Japan), and then placed onto a Kopf stereotaxic instrument. An L-shaped stainless steel 28-gauge cannula (Plastics One, Inc., USA) was bilaterally lowered into the hippocampus of each hemisphere using the following coordinates: anterior-posterior to the bregma = 3.3 mm; lateral = ±2.0 mm; ventral = 2.8 mm to the dura, respectively [19]. Cannulas were secured by dental cement and small screws. Silicone rubber tubes connected the cannulas to two Alzet minipumps (Model 1007D; pumping rate, 0.5 L/h for 7 days), which were implanted subcutaneously. 2.3.5. Histology At the end of the behavioral experiments, the rats were deeply anesthetized with sodium pentobarbital (120 mg/kg) and perfused with saline and ALTFiX® (FALMA, Japan) to fix their brain tissues. Brains were removed, sectioned in the coronal plane, and stained with cresyl violet. Sections were assessed under a
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3. Results 3.1. Histological results Fig. 2A shows a photomicrograph of a typical cannula track in a representative rat. The location of all injection cannula tips is indicated in Fig. 2B and C. All cannula tips were placed within the dorsal hippocampus.
3.2. Experiment 1: the effect of 5-day chronically infused d-AP5 on the 7-day retention of spatial reference memory 3.2.1. Training trials The mean escape latencies are shown in Fig. 3A. A two-way analysis of variance (ANOVA) with Group (aCSF, 15 mM d-AP5, and 30 mM d-AP5) as a between-subjects factor and Blocks (1–4) as a within-subject factor revealed only a significant main effect of Block (F(3,63) = 40.75, p < 0.001). Shaffer’s multiple comparison tests revealed that the latency of Block 1 significantly differed from those of Blocks 2–4 (p < 0.01).
Fig. 1. Schematic diagrams of the behavioral procedures. (A) Experiment 1: the retention interval was 7 days. Rats in each group received osmotic pump implantation (black arrow) 1 day after training trials (four trials/day for 4 days). Five days after this operation, the tubes that connected the cannulas to the pumps were cut (x-mark). (B) Experiment 2: the retention interval was 13 days. Imm- and Delgroups received osmotic pump implantation 1 day and 7 days after training trials, respectively.
bright-field microscope to determine whether the tip of the cannulas had entered the bilateral hippocampi.
2.4. Experiments 2.4.1. Experiment 1: the effect of 5-day chronically infused d-AP5 on the 7-day retention of spatial reference memory After finishing the training session, rats in each group received osmotic pump implantation 24 h after the last training day (Day 1). Five days after surgery (Day 6), tubes that connected the cannulas to the pumps were cut, and the chronic infusion of the drug was terminated. Seven days after the last training day, the probe test was conducted (Day 7) (Fig. 1A).
2.4.2. Experiment 2: the effect of immediate or delayed 5-day chronically infused d-AP5 on the 13-day retention of spatial reference memory Subjects were divided into Imm-aCSF, Imm-AP5, Del-aCSF and Del-AP5 groups in a manner similar to Experiment 1. Imm-groups underwent surgery 24 h after the last training day (Day 1), and the chronic infusion of the drug was terminated 5 days after the surgery (Day 6). Del-groups received surgery 7 days after the last training day (Day 7), and the chronic infusion of the drug was terminated 5 days after surgery (Day 12). Thirteen days after the last training day, the probe tests were conducted (Day 13) (Fig. 1A).
3.2.2. Probe test Fig. 4A shows the mean time spent in TQ. Both AP5 groups swam in the TQ significantly longer than the chance level (15 s) (15 mM: t(7) = 4.20, p = 0.002; 30 mM: t(7) = 2.15, p = 0.034), whereas the aCSF group did not (t(7) = 1.38, p = 0.105). Moreover, Dunnett’s multiple comparison tests revealed that the 15 mM d-AP5 group swam in TQ significantly longer than the aCSF group (p = 0.018), although the 30 mM d-AP5 group did not (p = 0.126). Additionally, both AP5 rats exhibited higher numbers of goal crossings than the controls did (Fig. 4C) as confirmed by Dunnett’s multiple comparison test (15 mM d-AP5: p = 0.005; 30 mM d-AP5: p = 0.040).
3.3. Experiment 2: the effect of immediate or delayed 5-day chronically infused d-AP5 on the 13-day retention of spatial reference memory 3.3.1. Training trials The mean escape latencies are shown in Fig. 3B. A three-way ANOVA with Phase (Imm and Del) and Drug (aCSF and AP5) as between-subjects factors and Blocks (1–4) as a within-subject factor revealed only a significant main effect of Block (F(3,78) = 47.2, p < 0.001). Shaffer’s multiple comparison tests revealed that Block 1 significantly differed from Block 2–4 (p < 0.01).
3.3.2. Probe test Fig. 4B shows the mean time spent in TQ. Only the Imm-AP5 group (t(7) = 2.98, p = 0.010) swam in the TQ significantly longer than the chance level (Imm-aCSF: t(6) = 0.17, p = 0.436; Del-aCSF: t(6) = 0.71, p = 0.253; Del-AP5: t(7) = 1.71, p = 0.065), although a twoway ANOVA with Phase and Drug as between-subjects factors revealed a non-significant main effects and interaction. Moreover, the Imm-AP5 rats exhibited higher numbers of goal crossings than the other groups did (Fig. 4D). This difference was confirmed by a two-way ANOVA with Phase and Drug as between-subjects factors, where a significant main effect of Drug (F(1,26) = 9.20, p = 0.005) and an interaction between the two factors (F(1,26) = 4.48, p = 0.044) were revealed. The simple main effect of drug was significant in the Imm-phase (F(1,26) = 13.26, p = 0.001) but not in the Del-phase (F(1,26) = 0.42, p = 0.523) group. In contrast, the simple main effect of Phase was not significant for either drug (aCSF: F(1,26) = 1.92, p = 0.178; AP5: F(1,26) = 2.62, p = 0.118).
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Fig. 2. Histology. (A) A photomicrograph of a typical cannula track in a representative rat. Scale bar = 1 mm. (B and C) The approximate locations of injection cannula tips in each group at Experiment 1 (B) and 2 (C). Anteroposterior distances (mm) to bregma are indicated beside each section.
3.4. Comparison between 15 mM d-AP5 infusion groups Next, we compared the three groups that were infused with 15 mM d-AP5. The 15 mM-AP5 group in Experiment 1 (Exp1-AP5) performed better during the probe test than the Imm-AP5 and Del-AP5 groups in Experiment 2. Regarding the time spent in TQ, a one-way ANOVA with these three groups as a betweensubject factor revealed a significant main effect (F(2,21) = 3.66, p = 0.043). Shaffer’s multiple comparison tests confirmed a significant difference between Exp1-AP5 and Del-AP5 (p = 0.013), but other comparisons did not. In contrast, concerning the number of goal crossings, Exp1-AP5 significantly differed from Imm-AP5 (p = 0.015) and Del-AP5 (p < 0.001), confirmed by Shaffer’s tests (main effect of groups using a one-way ANOVA was F(2,21) = 7.89,
Fig. 3. Acquisition performance in the training trials. Mean escape latencies during training trials in Experiment 1 (A) and 2 (B). Error bars represent SEMs.
p = 0.003). Because a high number of goal crossings indicates a successful navigation of the actual location of the escape platform, this measure is supposed to be a better index of the retention of spatial memory than the time spent in TQ. Therefore, on the basis of these comparisons, we suggest that memory retention in the Imm-AP5 rats declined in comparison with the Exp-AP5 group. 4. Discussion The aim of the present study was to assess whether a post-acquisition hippocampal NMDAR blockade suppresses the deterioration of spatial reference memory. All groups were able to reach similar levels of spatial reference memory after acquisition trainings in both Experiments 1 and 2, as evidenced by similar learning curves across groups and no significant behavioral differences between groups (see Fig. 3). In previous study from our lab [14] that used the same training procedure, there was an acquired reference memory for the platform location, and this lasted for at least 1 day. Therefore, it can be inferred from these results that the animals in the present study could acquire spatial reference memory. In the control groups, the acquired reference memory for location decreased to baseline levels within 7 days. These animals did not spend a significantly longer time in the TQ as compared to chance in the probe test conducted 7 or 13 days after training in Experiments 1 and 2 (see Fig. 4A and B). This is also consistent with our previous study demonstrating the decay of spatial reference memory in the control rats within 1 week of the post-acquisition period [14]. Our results suggest that blocking hippocampal NMDARs suppresses the deterioration of acquired reference memory for location. In Experiment 1 (7-day retention), rats infused with both
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Fig. 4. Retention performance in the probe test. (A and B) Mean time spent in the target and opposite quadrants in Experiment 1 (A) and 2 (B). The crossbar indicates the chance level. † p < 0.05 or †† p < 0.01 vs. chance level (15 s) by one-sample t test. # p < 0.05 vs. aCSF group by Dunnett’s multiple comparison test. (C and D) Mean number of goal point crossings in Experiment 1 (C) and 2 (D). # p < 0.05 or ## p < 0.01 vs. aCSF group by Dunnett’s multiple comparison test. ** p < 0.01; ANOVA followed by simple main effect tests. Error bars represent SEMs.
doses of d-AP5 following the training session had the highest performance in the probe test. Interestingly, in Experiment 2 (13-day retention), the immediate infusion of d-AP5 also facilitated the performance in a manner similar to Experiment 1, demonstrating that the retention of spatial memory persisted for 7 days after the termination of the NMDAR blockade. Based on the findings of Experiment 2, we can exclude at least two interpretations other than the suppressive effect of NMDAR blockade on memory decay. First, the rescued performance in the retention test by d-AP5 could not be caused by the inhibition of the retroactive interference with memory for location. If the new memory for experiences in the home cage (memory B) retroactively interfered with the memory for location (memory A), performance during the retention test (for memory A) would decrease in the control animals. During the retention phase, the chronically infused drug should have suppressed the acquisition of memory B, owing to the inhibitory function of hippocampal NMDARs in memory acquisition, and rescued the performance during the retention test. Therefore, the inhibition of this retroactive interference is one possible explanation of the drug effect. The retroactive interference, however, seems not to occur intrinsically in our experimental situation. If interference had occurred, then the performance of the aCSF group in Experiment 1 would have been similar to that of the Imm-AP5 group in Experiment
2. This is because the animals in these two groups experienced a period of drug-free phase (for 7 days); therefore the amounts of interference in their home cages during the retention phase were similar. This prediction contradicts our findings: the ImmAP5 group in Experiment 2 displayed the maintenance of spatial reference memory, whereas the aCSF group in Experiment 1 did not. This finding rejects the notion that retroactive interference, at least in our experimental situation, affected the performance at the retention test. Second, the rescued performance in the retention test by d-AP5 could not be caused by the inhibition of new memory acquisition during the retention test. In the retention probe test, animals would acquire novel information, for example, the platform is no longer there. If AP5 infusion was still effective on the day of the test and subsequently impaired the learning of the fact that the platform was no longer there, then animals might continue to search the location where the platform had been during training. However, in our experiments, the chronic infusion of d-AP5 was certainly terminated when the tubes connecting the cannulas to the pumps were cut. It has been reported that rats can acquire a spatial reference memory at a level similar to controls after the d-AP5 solution in an osmotic pump is exhausted [6]. Therefore, we can exclude the explanation that a preceding chronic infusion of d-AP5 inhibited the acquisition of a new memory.
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It should be noted that NMDAR blockade in the present study is supposed to not influence the initial consolidation of memory. We used the term “post-acquisition”, but not “post-training”, administration to describe our treatment. Typically, “post-training” treatment is given immediately after the end of training, which is assumed to influence the process of initial consolidation [20]. For example, Izquierdo and colleagues [21,22] have shown that hippocampal NMDAR blockade immediately, but not over 90 min, after training causes the impairment of memory retention in the inhibitory avoidance task. In the Morris water maze place task, similar amnesic effects have been reported with the immediate post-training administration of NMDAR antagonists [4,23–25]. In contrast, the chronic infusion of AP5 in the present study commenced 24 h after the end of training, that is to say, long after rats had acquired the task. Therefore, our finding indicates that the hippocampal NMDAR blockade facilitated the retention of the “acquired” (or consolidated) spatial reference memory. The present findings extend two particular points raised by previous studies. First, the present report clarifies that the dorsal hippocampus is an effective site for blocker injection. Intraventricular [14] and intraperitoneal [9] injections allow the drug to diffuse widely across the whole brain and, thus, it becomes difficult to specify its site of action. In fact, it has been confirmed that d-AP5 infused into the ventricle diffuses to other areas in addition to the hippocampus [6,26]. In contrast, we infused d-AP5 into the hippocampus and observed that it facilitated retention. An area-specific infusion of d-AP5 is important because the effects of the drug can differ depending on the infused area. For example, acquisition in the Morris water maze place task was impaired by intra-hippocampal, but not intra-amygdala, infusion of d-AP5 [4,27]. Our findings suggest that disturbing the function of the hippocampal NMDARs suppresses the deterioration of spatial reference memory. Second, the present data demonstrates that the facilitation of a post-acquisition NMDAR block can be observed not only in the radial maze task [9], but also in the Morris water maze task. The results from Villarreal et al. [9] and other studies [10–13] are conflicting. As mentioned in Section 1, we inferred that the level of maintained memory during the retention test in the control situation, rather than the differences in the properties inherent in these tasks, could explain the contradictory findings. We presumed that the memory of the control rats in the previous water maze studies had not already decayed back to the baseline level and, thus, the memory facilitation effect of the post-acquisition NMDAR blockade could not be detected. Here, we used a retention period that would cause the acquired spatial reference memory to decay back to baseline levels within 7 or 13 days in the control groups, as was the case with animals in the radial maze study [9]. Furthermore, we demonstrated that a post-acquisition NMDAR blockade suppresses the deterioration of spatial reference memory also in the Morris water maze task. Thus, our results increased the generality of the memory facilitation effect stemming from the use of NMDAR blockers after memory acquisition. Although the neural mechanisms by which hippocampal NMDAR blockade suppresses memory deterioration remain unclear, we speculate that sustained long-term potentiation (LTP) may underlie this phenomenon. LTP is thought to be a dominant form of neural plasticity and a biological basis of memory formation [28–30]. Several studies have reported that NMDA blockade can impair hippocampal LTP induction [3,31,32]. In contrast, Villarreal et al. [9] demonstrated that LTP was prolonged by post-induction CPP administration. Remarkably, several days after the last injection, the magnitude of hippocampal LTP decayed but was still higher than baseline or control levels. Our behavioral results from the current study are analogous to this electrophysiological data: in the Imm-AP5 group for Experiment 2, memory retention declined
(compared with 15 mM-AP5 group in Experiment 1) but remained at an elevated level (compared with controls) after the termination of d-AP5 infusion. In addition to sustained LTP, the inhibition of long-term depression (LTD) may be another neural substrate for the suppression of the spatial reference memory deterioration. Previous studies have reported that LTD induction was also inhibited by NMDAR blockade [33–35]. Notably, the administration of selective GluN2B NMDAR-subunit antagonists impaired the induction of LTD, but not LTP [36–42]. These antagonists also retarded the acquisition of new information during reversal learning in the water maze [37], which can be interpreted as the maintenance of previously acquired memory retention. Furthermore, some genetic [43,44] and pharmacological [45] approaches have demonstrated the important role of the hippocampal LTD in water-maze reversal learning. Although highly speculative, the facilitation of memory retention observed in our study may be caused by the inhibition of LTD induction, originating from the inactivation of GluN2B-containing NMDARs. However, the d-AP5 used in our study blocks all types of NMDARs, irrespective of subtype composition. Therefore, it is important for future studies to determine which subtypes of NMDAR blockers suppress the deterioration of spatial reference memory in a manner similar to the present study. In summary, we demonstrated that post-acquisition chronic blockade of hippocampal NMDARs for 5 days facilitates the 7-day or 13-day retention of acquired spatial reference memory. The memory, once it had deteriorated, was not enhanced by this treatment. These findings suggest that hippocampal NMDARs play an important role in the deterioration of spatial reference memory.
Acknowledgment We thank Dr. Tomoko Uekita for her technical guidance during the surgery.
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Research report
Post-acquisition hippocampal NMDA receptor blockade sustains retention of spatial reference memory in Morris water maze Keisuke Shinohara ∗ , Toshimichi Hata Faculty of Psychology, Doshisha University, Kyotanabe, Japan
h i g h l i g h t s • Post-acquisition hippocampal NMDAR blockade suppresses decay of spatial memory. • The memory, once it had deteriorated, was not enhanced by this treatment. • Hippocampal NMDARs are important for deterioration of spatial reference memory.
a r t i c l e
i n f o
Article history: Received 28 August 2013 Received in revised form 6 November 2013 Accepted 11 November 2013 Available online 17 November 2013 Keywords: Forgetting Spatial reference memory NMDA receptor Hippocampus
a b s t r a c t Several studies have demonstrated that the hippocampal N-methyl-d-aspartate type glutamate receptors (NMDARs) are necessary for the acquisition but not the retention of spatial reference memory. In contrast, a few studies have shown that post-acquisition repetitive intraperitoneal injections of an NMDAR antagonist facilitate the retention of spatial reference memory in a radial maze task. In the present study, we investigated the role of hippocampal NMDARs in the retention of spatial reference memories in Morris water maze. In Experiment 1, 24 h after training (4 trials/day for 4 days), d-AP5 was chronically infused into the hippocampus of rats for 5 days. In the subsequent probe test (seven days after training), we found that rats infused with d-AP5 spent a significantly longer time in the target quadrant compared to chance level, whereas rats in the control group did not. In Experiment 2, d-AP5 was infused into the hippocampus 1 (immediate) or 7 (delayed) days after the training session. In the probe test, following the retention interval of 13 days, immediate infusion facilitated the performance in a manner similar to Experiment 1, whereas the delayed infusion did not. These findings suggest that hippocampal NMDARs play an important role in the deterioration of spatial reference memory. © 2013 Elsevier B.V. All rights reserved.
1. Introduction A number of studies have demonstrated that the pharmacological blockade of N-methyl-d-aspartate type glutamate receptors (NMDARs) impairs the acquisition of spatial reference memory [1–8]. However, Villarreal et al. [9] demonstrate that the postacquisition administration of an NMDAR antagonist facilitated the retention of spatial reference memory. In their study, rats were trained in an 8-arm radial maze task (4 arms baited) and were then administrated repetitive intra-peritoneal injections of an NMDAR antagonist, (R,S)-3-(2-carboxypiperazin-4-yl) propyl1-phosphonic acid (CPP), during a 5-day retention period. In the subsequent retention test, fewer errors were committed by the CPP-treated rats than by the control rats. However, they did not
∗ Corresponding author at: Faculty of Psychology, Doshisha University, 1-3 TataraMiyakodani, Kyotanabe 610-0394, Japan. Tel.: +81 0774 65 7087; fax: +81 0774 65 7250. E-mail address: [email protected] (K. Shinohara). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.11.016
specify whether the effect was present in working or reference memory, because the performance in this task is dependent on both types of memory. To our knowledge, only few studies have supported the findings of the previously mentioned study [9], using tasks that are thought to primarily involve spatial reference memory. Namely, blocking NMDARs after the acquisition of spatial reference memory does not affect retention [10–13]. For example, in the Morris water maze place task, an NMDAR antagonist, d-2-amino-5-phosphonovaleric acid (d-AP5), was chronically infused into the lateral ventricle for 7 days following training. Treated rats performed similarly to controls on a retention probe test conducted 14 days after training [11]. This result seems to conflict with that of Villarreal et al. [9]. However, these studies cannot be compared, because there was at least one major methodological difference between them. The key variable may be the retention interval period. The retention interval period mentioned in Villarreal et al. [9] was such that the memories of the control group at the time of the retention test had already decayed back to the baseline level. However, other studies [10–13] reported that the memory was still
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above the baseline. In the latter cases, even if the post-acquisition NMDAR blockade had memory facilitation effects, it might have been masked by a ceiling effect. However, the study by Villarreal et al. [9] allows the detection of any possible memory facilitation effects of NMDAR antagonists. Therefore, the findings of Villarreal et al. [9] and other study findings [10–13] cannot be compared. In light of this unresolved issue in the literature, we previously used a Morris water maze to examine the effect of post-acquisition NMDAR blocking on retention test performance with respect to spatial memory, which persisted for 1 day but decayed within 7 days [14]. In such a situation, we demonstrated that chronic infusion of d-AP5 in the lateral ventricle post-acquisition maintained the memory for platform location in the probe test at seven days after training. This finding suggested that memory facilitation effects of the post-acquisition NMDAR blockade can be observed when the acquired memory has decayed in normal animals. Which brain structure functions as the critical site for the facilitating effect of NMDAR blockers? The hippocampus is a predominant candidate, since it has been closely involved in the acquisition of spatial reference memory [15,16]. However, the sites that are affected by NMDAR blocking have not yet been confirmed. This is because in studies that have reported memory facilitation effects, the NMDAR blockers were infused intraperitoneally [9] or intraventricularly [14]; therefore, affecting NMDARs throughout the whole brain. In the present study, we chronically infused d-AP5 into the dorsal hippocampus to elucidate the role of hippocampal NMDAR in the deterioration of spatial reference memory after its acquisition. We demonstrate, for the first time, that the chronic infusion of d-AP5 into the dorsal hippocampus after training suppresses the deterioration of spatial reference memory. These findings suggest that hippocampal NMDARs play an important role in the decay of spatial reference memory.
2. Materials and methods
2.2. Apparatus A circular pool, 140 cm in diameter with a 50-cm-high wall, was used. Both the pool floor and wall were black. The pool was filled with water (24 ± 1 ◦ C) to a depth of 13 cm and made opaque by black carbon ink. A number of environmental cues were present in the experimental room. The escape platform was a black cylinder 12 cm in diameter and 11 cm in height. It was submerged 2 cm below the water surface in the center of a specific quadrant. This shallower pool, compared with other water maze studies [17], can be used as a tool to assess rat behavior in the water maze because male adult Wistar rats can successfully remember the location of the platform at a depth of around 10 cm [18]. The shallow depth of the water maze has an advantage over a deep one in that the quality and temperature of the water can be managed more easily. 2.3. General behavioral procedures 2.3.1. Training session Following handling (5 min/day for 5 days), rats were trained in four trials (with at least a 10-min inter-trial interval) per day for 4 days. In the acquisition training trials, the location of the submerged escape platform was fixed at the center of a quadrant (target quadrant; TQ) for each rat. The rat was placed in the water facing the wall of the pool at one of the four starting points (north, east, south, or west). If the rat could not find the platform within 60 s, it was gently guided to the platform by the experimenter. The rat was left on the platform for 15 s and then removed from the pool and dried with a towel. 2.3.2. Probe test After a retention interval of 7 or 13 days, a probe test was conducted. In the probe test, the rats were allowed to swim freely without the platform for 60 s. The starting position was the point opposite the target. The time spent in TQ and the number of goal crossings (traversing the actual location of the escape platform) were recorded.
2.1. Subjects Fifty-eight naïve male albino Wistar rats (Shimizu, Kyoto, Japan) were used for this experiment; however, four rats were excluded from the analysis as their hippocampi were injured due to chronic infusion. At the beginning of training, all rats were 10–11 weeks old. Rats were housed individually with food and water available ad libitum and a controlled 12-h light–dark cycle (from 8:00 AM to 8:00 PM). Experiments were conducted during the light period (from 10:00 AM to 2:00 PM). All procedures and treatments were approved by the Doshisha Committee of Animal Experiment. In Experiment 1, 24 rats were divided into three groups, artificial cerebral spinal fluid (aCSF) infused (aCSF; n = 8), 15 mM d-AP5infused (n = 8), or 30 mM d-AP5-infused (n = 8), groups in such a way that the mean escape latencies in the last training day were counterbalanced. The average body weight (mean ± standard deviation [SD]) of the aCSF, 15 mM d-AP5, and 30 mM d-AP5 groups were 276.8 ± 14.9 g, 273.8 ± 15.8 g, and 274.9 ± 21.4 g, respectively. In Experiment 2, 30 rats were divided into an immediate aCSF-infused (Imm-aCSF; n = 7), an immediate 15 mM d-AP5-infused (Imm-AP5; n = 8), a delayed aCSF-infused (DelaCSF; n = 7), and a delayed 15 mM d-AP5-infused (Del-AP5; n = 8) group. The average body weight (mean ± SD) of the Imm-aCSF, Imm-AP5, Del-aCSF, and Del-AP5 groups were 273.4 ± 13.7 g, 265.5 ± 10.7 g, 273.4 ± 10.4 g, and 276.8 ± 11.5 g, respectively.
2.3.3. Drug preparation d-AP5 (Enzo Life Sciences, Inc., USA) was dissolved in aCSF and 15 mM and 30 mM solutions were prepared. Before implantation, minipumps were filled with the solution and incubated at 37 ◦ C for 24 h for priming and stabilization of the flow. 2.3.4. Surgery Rats received an intraperitoneal injection of atropine sulfate (0.1 mL; Tanabe Seiyaku, Japan), were anesthetized with sodium pentobarbital (60 mg/kg; Kyoritsu Seiyaku, Japan), and then placed onto a Kopf stereotaxic instrument. An L-shaped stainless steel 28-gauge cannula (Plastics One, Inc., USA) was bilaterally lowered into the hippocampus of each hemisphere using the following coordinates: anterior-posterior to the bregma = 3.3 mm; lateral = ±2.0 mm; ventral = 2.8 mm to the dura, respectively [19]. Cannulas were secured by dental cement and small screws. Silicone rubber tubes connected the cannulas to two Alzet minipumps (Model 1007D; pumping rate, 0.5 L/h for 7 days), which were implanted subcutaneously. 2.3.5. Histology At the end of the behavioral experiments, the rats were deeply anesthetized with sodium pentobarbital (120 mg/kg) and perfused with saline and ALTFiX® (FALMA, Japan) to fix their brain tissues. Brains were removed, sectioned in the coronal plane, and stained with cresyl violet. Sections were assessed under a
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3. Results 3.1. Histological results Fig. 2A shows a photomicrograph of a typical cannula track in a representative rat. The location of all injection cannula tips is indicated in Fig. 2B and C. All cannula tips were placed within the dorsal hippocampus.
3.2. Experiment 1: the effect of 5-day chronically infused d-AP5 on the 7-day retention of spatial reference memory 3.2.1. Training trials The mean escape latencies are shown in Fig. 3A. A two-way analysis of variance (ANOVA) with Group (aCSF, 15 mM d-AP5, and 30 mM d-AP5) as a between-subjects factor and Blocks (1–4) as a within-subject factor revealed only a significant main effect of Block (F(3,63) = 40.75, p < 0.001). Shaffer’s multiple comparison tests revealed that the latency of Block 1 significantly differed from those of Blocks 2–4 (p < 0.01).
Fig. 1. Schematic diagrams of the behavioral procedures. (A) Experiment 1: the retention interval was 7 days. Rats in each group received osmotic pump implantation (black arrow) 1 day after training trials (four trials/day for 4 days). Five days after this operation, the tubes that connected the cannulas to the pumps were cut (x-mark). (B) Experiment 2: the retention interval was 13 days. Imm- and Delgroups received osmotic pump implantation 1 day and 7 days after training trials, respectively.
bright-field microscope to determine whether the tip of the cannulas had entered the bilateral hippocampi.
2.4. Experiments 2.4.1. Experiment 1: the effect of 5-day chronically infused d-AP5 on the 7-day retention of spatial reference memory After finishing the training session, rats in each group received osmotic pump implantation 24 h after the last training day (Day 1). Five days after surgery (Day 6), tubes that connected the cannulas to the pumps were cut, and the chronic infusion of the drug was terminated. Seven days after the last training day, the probe test was conducted (Day 7) (Fig. 1A).
2.4.2. Experiment 2: the effect of immediate or delayed 5-day chronically infused d-AP5 on the 13-day retention of spatial reference memory Subjects were divided into Imm-aCSF, Imm-AP5, Del-aCSF and Del-AP5 groups in a manner similar to Experiment 1. Imm-groups underwent surgery 24 h after the last training day (Day 1), and the chronic infusion of the drug was terminated 5 days after the surgery (Day 6). Del-groups received surgery 7 days after the last training day (Day 7), and the chronic infusion of the drug was terminated 5 days after surgery (Day 12). Thirteen days after the last training day, the probe tests were conducted (Day 13) (Fig. 1A).
3.2.2. Probe test Fig. 4A shows the mean time spent in TQ. Both AP5 groups swam in the TQ significantly longer than the chance level (15 s) (15 mM: t(7) = 4.20, p = 0.002; 30 mM: t(7) = 2.15, p = 0.034), whereas the aCSF group did not (t(7) = 1.38, p = 0.105). Moreover, Dunnett’s multiple comparison tests revealed that the 15 mM d-AP5 group swam in TQ significantly longer than the aCSF group (p = 0.018), although the 30 mM d-AP5 group did not (p = 0.126). Additionally, both AP5 rats exhibited higher numbers of goal crossings than the controls did (Fig. 4C) as confirmed by Dunnett’s multiple comparison test (15 mM d-AP5: p = 0.005; 30 mM d-AP5: p = 0.040).
3.3. Experiment 2: the effect of immediate or delayed 5-day chronically infused d-AP5 on the 13-day retention of spatial reference memory 3.3.1. Training trials The mean escape latencies are shown in Fig. 3B. A three-way ANOVA with Phase (Imm and Del) and Drug (aCSF and AP5) as between-subjects factors and Blocks (1–4) as a within-subject factor revealed only a significant main effect of Block (F(3,78) = 47.2, p < 0.001). Shaffer’s multiple comparison tests revealed that Block 1 significantly differed from Block 2–4 (p < 0.01).
3.3.2. Probe test Fig. 4B shows the mean time spent in TQ. Only the Imm-AP5 group (t(7) = 2.98, p = 0.010) swam in the TQ significantly longer than the chance level (Imm-aCSF: t(6) = 0.17, p = 0.436; Del-aCSF: t(6) = 0.71, p = 0.253; Del-AP5: t(7) = 1.71, p = 0.065), although a twoway ANOVA with Phase and Drug as between-subjects factors revealed a non-significant main effects and interaction. Moreover, the Imm-AP5 rats exhibited higher numbers of goal crossings than the other groups did (Fig. 4D). This difference was confirmed by a two-way ANOVA with Phase and Drug as between-subjects factors, where a significant main effect of Drug (F(1,26) = 9.20, p = 0.005) and an interaction between the two factors (F(1,26) = 4.48, p = 0.044) were revealed. The simple main effect of drug was significant in the Imm-phase (F(1,26) = 13.26, p = 0.001) but not in the Del-phase (F(1,26) = 0.42, p = 0.523) group. In contrast, the simple main effect of Phase was not significant for either drug (aCSF: F(1,26) = 1.92, p = 0.178; AP5: F(1,26) = 2.62, p = 0.118).
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Fig. 2. Histology. (A) A photomicrograph of a typical cannula track in a representative rat. Scale bar = 1 mm. (B and C) The approximate locations of injection cannula tips in each group at Experiment 1 (B) and 2 (C). Anteroposterior distances (mm) to bregma are indicated beside each section.
3.4. Comparison between 15 mM d-AP5 infusion groups Next, we compared the three groups that were infused with 15 mM d-AP5. The 15 mM-AP5 group in Experiment 1 (Exp1-AP5) performed better during the probe test than the Imm-AP5 and Del-AP5 groups in Experiment 2. Regarding the time spent in TQ, a one-way ANOVA with these three groups as a betweensubject factor revealed a significant main effect (F(2,21) = 3.66, p = 0.043). Shaffer’s multiple comparison tests confirmed a significant difference between Exp1-AP5 and Del-AP5 (p = 0.013), but other comparisons did not. In contrast, concerning the number of goal crossings, Exp1-AP5 significantly differed from Imm-AP5 (p = 0.015) and Del-AP5 (p < 0.001), confirmed by Shaffer’s tests (main effect of groups using a one-way ANOVA was F(2,21) = 7.89,
Fig. 3. Acquisition performance in the training trials. Mean escape latencies during training trials in Experiment 1 (A) and 2 (B). Error bars represent SEMs.
p = 0.003). Because a high number of goal crossings indicates a successful navigation of the actual location of the escape platform, this measure is supposed to be a better index of the retention of spatial memory than the time spent in TQ. Therefore, on the basis of these comparisons, we suggest that memory retention in the Imm-AP5 rats declined in comparison with the Exp-AP5 group. 4. Discussion The aim of the present study was to assess whether a post-acquisition hippocampal NMDAR blockade suppresses the deterioration of spatial reference memory. All groups were able to reach similar levels of spatial reference memory after acquisition trainings in both Experiments 1 and 2, as evidenced by similar learning curves across groups and no significant behavioral differences between groups (see Fig. 3). In previous study from our lab [14] that used the same training procedure, there was an acquired reference memory for the platform location, and this lasted for at least 1 day. Therefore, it can be inferred from these results that the animals in the present study could acquire spatial reference memory. In the control groups, the acquired reference memory for location decreased to baseline levels within 7 days. These animals did not spend a significantly longer time in the TQ as compared to chance in the probe test conducted 7 or 13 days after training in Experiments 1 and 2 (see Fig. 4A and B). This is also consistent with our previous study demonstrating the decay of spatial reference memory in the control rats within 1 week of the post-acquisition period [14]. Our results suggest that blocking hippocampal NMDARs suppresses the deterioration of acquired reference memory for location. In Experiment 1 (7-day retention), rats infused with both
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Fig. 4. Retention performance in the probe test. (A and B) Mean time spent in the target and opposite quadrants in Experiment 1 (A) and 2 (B). The crossbar indicates the chance level. † p < 0.05 or †† p < 0.01 vs. chance level (15 s) by one-sample t test. # p < 0.05 vs. aCSF group by Dunnett’s multiple comparison test. (C and D) Mean number of goal point crossings in Experiment 1 (C) and 2 (D). # p < 0.05 or ## p < 0.01 vs. aCSF group by Dunnett’s multiple comparison test. ** p < 0.01; ANOVA followed by simple main effect tests. Error bars represent SEMs.
doses of d-AP5 following the training session had the highest performance in the probe test. Interestingly, in Experiment 2 (13-day retention), the immediate infusion of d-AP5 also facilitated the performance in a manner similar to Experiment 1, demonstrating that the retention of spatial memory persisted for 7 days after the termination of the NMDAR blockade. Based on the findings of Experiment 2, we can exclude at least two interpretations other than the suppressive effect of NMDAR blockade on memory decay. First, the rescued performance in the retention test by d-AP5 could not be caused by the inhibition of the retroactive interference with memory for location. If the new memory for experiences in the home cage (memory B) retroactively interfered with the memory for location (memory A), performance during the retention test (for memory A) would decrease in the control animals. During the retention phase, the chronically infused drug should have suppressed the acquisition of memory B, owing to the inhibitory function of hippocampal NMDARs in memory acquisition, and rescued the performance during the retention test. Therefore, the inhibition of this retroactive interference is one possible explanation of the drug effect. The retroactive interference, however, seems not to occur intrinsically in our experimental situation. If interference had occurred, then the performance of the aCSF group in Experiment 1 would have been similar to that of the Imm-AP5 group in Experiment
2. This is because the animals in these two groups experienced a period of drug-free phase (for 7 days); therefore the amounts of interference in their home cages during the retention phase were similar. This prediction contradicts our findings: the ImmAP5 group in Experiment 2 displayed the maintenance of spatial reference memory, whereas the aCSF group in Experiment 1 did not. This finding rejects the notion that retroactive interference, at least in our experimental situation, affected the performance at the retention test. Second, the rescued performance in the retention test by d-AP5 could not be caused by the inhibition of new memory acquisition during the retention test. In the retention probe test, animals would acquire novel information, for example, the platform is no longer there. If AP5 infusion was still effective on the day of the test and subsequently impaired the learning of the fact that the platform was no longer there, then animals might continue to search the location where the platform had been during training. However, in our experiments, the chronic infusion of d-AP5 was certainly terminated when the tubes connecting the cannulas to the pumps were cut. It has been reported that rats can acquire a spatial reference memory at a level similar to controls after the d-AP5 solution in an osmotic pump is exhausted [6]. Therefore, we can exclude the explanation that a preceding chronic infusion of d-AP5 inhibited the acquisition of a new memory.
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It should be noted that NMDAR blockade in the present study is supposed to not influence the initial consolidation of memory. We used the term “post-acquisition”, but not “post-training”, administration to describe our treatment. Typically, “post-training” treatment is given immediately after the end of training, which is assumed to influence the process of initial consolidation [20]. For example, Izquierdo and colleagues [21,22] have shown that hippocampal NMDAR blockade immediately, but not over 90 min, after training causes the impairment of memory retention in the inhibitory avoidance task. In the Morris water maze place task, similar amnesic effects have been reported with the immediate post-training administration of NMDAR antagonists [4,23–25]. In contrast, the chronic infusion of AP5 in the present study commenced 24 h after the end of training, that is to say, long after rats had acquired the task. Therefore, our finding indicates that the hippocampal NMDAR blockade facilitated the retention of the “acquired” (or consolidated) spatial reference memory. The present findings extend two particular points raised by previous studies. First, the present report clarifies that the dorsal hippocampus is an effective site for blocker injection. Intraventricular [14] and intraperitoneal [9] injections allow the drug to diffuse widely across the whole brain and, thus, it becomes difficult to specify its site of action. In fact, it has been confirmed that d-AP5 infused into the ventricle diffuses to other areas in addition to the hippocampus [6,26]. In contrast, we infused d-AP5 into the hippocampus and observed that it facilitated retention. An area-specific infusion of d-AP5 is important because the effects of the drug can differ depending on the infused area. For example, acquisition in the Morris water maze place task was impaired by intra-hippocampal, but not intra-amygdala, infusion of d-AP5 [4,27]. Our findings suggest that disturbing the function of the hippocampal NMDARs suppresses the deterioration of spatial reference memory. Second, the present data demonstrates that the facilitation of a post-acquisition NMDAR block can be observed not only in the radial maze task [9], but also in the Morris water maze task. The results from Villarreal et al. [9] and other studies [10–13] are conflicting. As mentioned in Section 1, we inferred that the level of maintained memory during the retention test in the control situation, rather than the differences in the properties inherent in these tasks, could explain the contradictory findings. We presumed that the memory of the control rats in the previous water maze studies had not already decayed back to the baseline level and, thus, the memory facilitation effect of the post-acquisition NMDAR blockade could not be detected. Here, we used a retention period that would cause the acquired spatial reference memory to decay back to baseline levels within 7 or 13 days in the control groups, as was the case with animals in the radial maze study [9]. Furthermore, we demonstrated that a post-acquisition NMDAR blockade suppresses the deterioration of spatial reference memory also in the Morris water maze task. Thus, our results increased the generality of the memory facilitation effect stemming from the use of NMDAR blockers after memory acquisition. Although the neural mechanisms by which hippocampal NMDAR blockade suppresses memory deterioration remain unclear, we speculate that sustained long-term potentiation (LTP) may underlie this phenomenon. LTP is thought to be a dominant form of neural plasticity and a biological basis of memory formation [28–30]. Several studies have reported that NMDA blockade can impair hippocampal LTP induction [3,31,32]. In contrast, Villarreal et al. [9] demonstrated that LTP was prolonged by post-induction CPP administration. Remarkably, several days after the last injection, the magnitude of hippocampal LTP decayed but was still higher than baseline or control levels. Our behavioral results from the current study are analogous to this electrophysiological data: in the Imm-AP5 group for Experiment 2, memory retention declined
(compared with 15 mM-AP5 group in Experiment 1) but remained at an elevated level (compared with controls) after the termination of d-AP5 infusion. In addition to sustained LTP, the inhibition of long-term depression (LTD) may be another neural substrate for the suppression of the spatial reference memory deterioration. Previous studies have reported that LTD induction was also inhibited by NMDAR blockade [33–35]. Notably, the administration of selective GluN2B NMDAR-subunit antagonists impaired the induction of LTD, but not LTP [36–42]. These antagonists also retarded the acquisition of new information during reversal learning in the water maze [37], which can be interpreted as the maintenance of previously acquired memory retention. Furthermore, some genetic [43,44] and pharmacological [45] approaches have demonstrated the important role of the hippocampal LTD in water-maze reversal learning. Although highly speculative, the facilitation of memory retention observed in our study may be caused by the inhibition of LTD induction, originating from the inactivation of GluN2B-containing NMDARs. However, the d-AP5 used in our study blocks all types of NMDARs, irrespective of subtype composition. Therefore, it is important for future studies to determine which subtypes of NMDAR blockers suppress the deterioration of spatial reference memory in a manner similar to the present study. In summary, we demonstrated that post-acquisition chronic blockade of hippocampal NMDARs for 5 days facilitates the 7-day or 13-day retention of acquired spatial reference memory. The memory, once it had deteriorated, was not enhanced by this treatment. These findings suggest that hippocampal NMDARs play an important role in the deterioration of spatial reference memory.
Acknowledgment We thank Dr. Tomoko Uekita for her technical guidance during the surgery.
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