When do rats start to forget

In order to determine the time course of forgetting, animals were trained in an object location (OL) task. Animals were presented with two identical copies of a junk object (metal can) located at stable positions in a familiar open field. Memory was assessed 1, 3, 5 or 7 days after training. To this end, one of the two objects was moved to a new location, and relative preference to explore this novelty was assessed as indicating long-term object location memory. The novelty preference indices for all groups and the total exploration time are shown in Fig. 1. We found that animals tested 1 or 3 days after training expressed a significant preference to the displaced object (t(4) = −3.29; p = 0.03; t(5) = −2.98; p = 0.03, paired t-test comparing exploratory preference during training and memory test). However, animals tested 5 or 7 days after training showed no preference for the displaced object (t(4) = 0.83; p = 0.44; t(4) = −1.58; p = 0.18, paired t-test), indicating that spatial memory was forgotten. The overall time spent exploring the objects was not different among the groups as revealed by one way ANOVA (F(3, 40) = 2.21; p = 0.10; Fig. 1B).

Figure 1: Rats maintain the object location memory in 1 and 3, but not in 5 or 7 days after learning. (A) Experimental design of the OL paradigm (top panel), and the exploration ratios during the training (white bars) and retention tests (blue bars) performed on days 1, 3, 5, or 7 after learning. Rats keep their memory until day 3, since they spent more time exploring the object that was switched into a new position. (B) The overall exploration activity in the test does not differ among all groups. Dotted line indicates similar exploration activity between both novel and familiar objects. Data expressed as mean ± SEM. *p < 0.5. All other comparisons were not significant (n = 4 to 6 per group). Full size image

Thus, forgetting of OL memory starts sometime between 3 and 5 days after training in the present protocol.

Role of NMDAR in forgetting: chronic systemic injections of NMDAR antagonists maintain long-term memory

It has been previously shown that chronic NMDAR blockade can sustain spatial memory in the radial-arm maze6 as well as in the Morris water maze7, and that it blocks the decay of LTP in freely moving animals6. To explore whether this is also the case for the OL task, rats were tested in a time-point when this type of memory would normally be forgotten. After the training session, we injected systemically the NMDAR antagonist memantine (10 or 20 mg/kg) or its vehicle once a day, with the first injection 6 h after the last day of training (Fig. 2A). During the drug-free retention test performed 7 d after training, a one-way ANOVA detected a significant difference among the groups in the exploration of the displaced object (F(2, 17) = 5.10; p = 0.01). Post hoc comparisons revealed that the group treated with 20 mg/kg of memantine explored the displaced object significantly longer than the control group (p = 0.01). Additionally, both vehicle and 10 mg/kg memantine-treated groups expressed no preference for the object at the novel location compared to exploration during training (t(5) = −0.84; p = 0.43; t(6) = −1.81; p = 0.11, paired t-tests). However, the group treated with 20 mg/kg of memantine preferred exploring the displaced object (t(5) = −3.41; p = 0.01, paired t-test). The overall time spent exploring the objects was not different among the groups, as revealed by one-way ANOVA (F(2, 33) = 2.25; p = 0.12; Fig. 2D, left panel). Moreover, to determine whether this effect could have been caused by an early NMDAR blockade (around 6–12 h after training), we trained another set of animals and tested them 7 d later. In contrast to the previous experiments, however, these animals received a single injection of 20 mg/kg of memantine or vehicle 6 h after training, and then only vehicle following the 6 day retention interval. Both groups expressed no preference for the novel-located object in the test (mean + SEM exploration of the displaced object in the training: vehicle = 44.56 ± 3.82; memantine = 49.68 ± 3.21, and test: vehicle = 51.78 ± 1, 87; memantine = 56.39 ± 3.21; t(5) = −1.52; p = 0.18; t(5) = −1.89; p = 0.18; paired t-test; n = 6 per group). There were no differences between the groups (t(10) = 1.24; p = 0.24; unpaired t-test), indicating that LTM maintenance over time is not determined by early NMDAR blockade (data not shown).

Figure 2: Long-term memory forgetting is regulated through NMDAR-dependent signaling. (A) Dose-dependent effect of the NMDAR antagonist memantine upon the test conducted 7 days after training in the OL task. Rats chronically treated with the high dose of memantine explored significantly more the displaced object compared to other groups. (B) The NMDAR antagonist MK801 prevent memory forgetting for either 7 or 10 days after learning in the same paradigm. (C) NMDAR inhibition also regulates the decay of the OR memory. White bars represent the performance in the training, and blue bars in the tests. (D) The overall exploration does not differ among the groups, indicating that memantine or MK801 have no effect on basal exploratory activity. Data expressed as mean ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001 (n = 6 to 9 per group). Full size image

In order to rule out the possibility of a non-specific memantine effect on forgetting, we performed the same experiment with another set of animals injecting every 12 h the potent NMDAR antagonist MK801. The MK801-treated group, but not the vehicle group, preferred exploring the displaced object in the test performed on day 7 compared with the training (t(8) = −9.29; p = 0.00001; t(5) = −0.98; p = 0.36, paired t-test; Fig. 2B). An additional set of animals kept receiving MK801 and was tested for memory retention on day 10. Interestingly, these animals were able to maintain the preference for the novel location object even at this time-point after training (t(6) = −5.52; p = 0.001, paired t-test), indicating that forgetting of spatial LTM is mediated through NMDAR-dependent signaling. Further analysis revealed that animals chronically treated with MK801 (for 7 or 10 d) significantly spent more time exploring the new object location than the control group (one-way ANOVA, F(2, 19) = 13.078; p = 0.0002, followed by SNK post hoc analysis, control group vs MK801 7 d, p = 0.0003, and control vs MK801 10 d, p = 0.03, respectively; Fig. 2C). Also, the groups did not differ in total exploration time (one-way ANOVA, F(2, 43) = 1.50; p = 0.23; Fig. 2D, middle panel), revealing that the treatment do not alter basal exploration activity.

Most of the studies regarding the mechanisms of forgetting (as well as the above results) were performed in hippocampal-dependent tasks6,7,25. We therefore next asked if the same mechanisms would be involved in a task that does not require the hippocampus, such as object recognition (OR) paradigm26. In this case, instead of moving an object to a new location during the test, one of the familiar objects was replaced with a new one and remained at its original location. Animals were trained in the OR task and tested one week later for memory retention (Fig. 2C). As in the above experiments, we injected systemically the NMDAR antagonist memantine (20 mg/kg) or its vehicle once a day between training and test. The control group expressed no preference for the novel object in the test (t(5) = 1, 07; p = 0.33, paired t-test), suggesting that object recognition memory was forgotten at this point in time. However, the memantine 20 mg/kg-treated group preferred exploring the new object to the old one presented in the training (t(7) = −3, 87; p = 0.006, paired t-test). Further, the group treated with memantine showed enhanced OR memory 7 d after training compared to the control group (t(12) = 2, 23; p = 0.04, unpaired t-test). As expected, the overall time spent exploring the objects was not different between the groups (t(26) = 1.14; p = 0.26, unpaired t-test; Fig. 2D, right panel). These data suggest that NMDAR activation is involved in forgetting of memories that are not maintained in the hippocampus26.

Taken together, these results provide additional support for the notion that forgetting requires NMDAR-mediated signaling.

GluN2B-containing NMDAR regulates LTP decay in the hippocampus

NMDAR are heterotetramers. In the mammalian brain, most NMDARs are comprised of either GluN1 and GluN2A or GluN1 and GluN2B. Yashiro and Philpot27 have shown that, depending on the NMDAR subunit composition, its activation may induce distinct plasticity patterns, such as LTD or LTP27. The GluN2B-containing NMDAR enables a higher Ca2+ influx into postsynaptic neurons, and has been associated with plastic events such as fear memory destabilization28 and memory updating23,24. Recently it has been suggested that the GluN2B might be critical in regulating forgetting4,5. Indeed, strong emotional memories that are not forgotten have been associated with downregulation of GluN2B10.

To address whether GluN2B-NMDARs would be involved in the decay of hippocampal LTP, we used a protocol that induces LTP decaying within 90 min after the HFS25. In this case, thirty minutes after administering HFS in anesthetized animals, we infused the GluN2B-NMDAR antagonist ifenprodil or its vehicle in the CA1 region of the hippocampus. This protocol induced LTP in both the ifenprodil-treated group (206.6% ± 1.35 at 50 min after the HFS; p = 0.014 vs baseline; repeated measures ANOVA followed by SNK post hoc test) and in the control group (207.6% ± 1.36; p = 0.003 vs baseline; repeated measures ANOVA followed by SNK post hoc). Nonetheless, 180 min after the HFS, in the control group, LTP has decayed back to basal levels (99.2% ± 3.25; p = 0.972 vs baseline; repeated measures ANOVA followed by SNK post hoc test). However, intrahippocampal infusions of ifenprodil induced a nondecaying LTP (170.36% ± 4.20; p = 0.03 vs baseline; repeated measures ANOVA followed by SNK; Fig. 3). Further analysis revealed that the ifenprodil-treated group presented a higher amplitude 180 min after the HFS comparing with their relative controls (t(6) = 3.03; p = 0.02, independent t-test), but not 50 min after the HFS (t(6) = −0.02; p = 0.98, independent t-test). In addition, we performed the same experiment as described above using a lower ifenprodil concentration (0.1 μg/μL). No difference was found in the 10 min prior to HFS, t50–60 min or t170–180 min (p > 0.05, data not shown). An additional experiment was performed in order to test whether ifenprodil affects basal transmission in the hippocampus. No difference was found between ifenprodil and vehicle groups (data not shown).

Figure 3: Hippocampal LTP decay in vivo is regulated by GluN2B-NMDAR. Corresponding representative traces of anesthetized rats infused with vehicle or ifenprodil (GluN2B-NMDAR antagonist) 30 min after LTP induction. The plot presents CA1-evoked synaptic potentials recorded for 180 min (left panel). In this protocol, hippocampal LTP in the control group start to decay around 90 min after HFS, whereas the application of the GluN2B-NMDAR antagonist prevents the natural decay of LTP. Right panel shown the% of change of normalized amplitude in three distinct intervals (1 = baseline response; 2 = 50–60 min after HFS; 3 = 170–180 min after HFS) (n = 4 per group). Full size image

Taken together, these data show that GluN2B-containing NMDAR is robustly engaged in the decay of short-lasting LTP in the hippocampus.

Ca2+-dependent signaling is essential to regulate spatial memory maintenance: role of LVDCCs

The mechanisms that promote forgetting of LTM may engage various Ca2+ signaling pathways. For example, the L-type voltage dependent calcium channel (LVDCC) has been associated with memory destabilization required during memory updating12,13,23,24. To address whether LVDCC activation is also involved in forgetting, we trained animals in the OL paradigm, as described above. Animals were systemically treated with the LVDCC inhibitor nimodipine or its vehicle once per day, and tested 7 d after training. As shown in Fig. 4A, nimodipine-treated animals exhibited enhanced OL memory when tested 7 d after training (t(10) = 2, 65; p = 0.02, unpaired t-test). As expected, the control group expressed no preference for the novel-located object in the test (t(5) = −1.56; p = 0.17, paired t-test). However, rats treated with nimodipine preferred the displaced object to the object at the old location (t(5) = −4, 30; p = 0.007, paired t-test). Both groups did not differ in total exploration time (t(22) = −0.29; p = 0.77, unpaired t-test; Fig. 4B), suggesting that the treatment did not alter basal exploratory activity.

Figure 4: Ca2+ -dependent signaling through LVDCCs regulates long-term memory maintenance. (A) Rats treated chronically with nimodipine, a LVDCC blocker, significantly spent more time exploring the new object location than the control group during the test conducted 7 d after training. White bars represent the performance in the training, and blue bars in the test. (B) The overall exploration does not differ between the groups. Data expressed as mean ± SEM. *p < 0.5, **p < 0.01 (n = 6 per group). Full size image

In conclusion, our results show for the first time that forgetting of long-term memory depends on the Ca2+ influx through LVDCC.

Calcineurin positively modulates long-term memory forgetting

The above experiments have shown that the Ca2+ influx through both LVDCCs and NMDARs regulates forgetting of object location memory. Next, we investigated possible signaling pathways activated by the Ca2+ influx. Phosphatases are thought to negatively regulate synaptic plasticity and memory15,16,17,18,19. The phosphatase CaN is rapidly activated by Ca2+ influx29, requiring both NMDAR and LVDCC activation30,31. We predicted that if the Ca2+ influx activates CaN in order to induce forgetting, then inhibiting CaN activity would maintain LTM in a nondecaying state. To test this hypothesis, rats were trained in the OL task, chronically treated with the CaN inhibitor FK506 or their vehicle, and tested 7 d later. As shown in Fig. 5A, both groups preferred the displaced object to the object at the old location in the test (t(7) = −3, 33; p = 0.013 and t(8) = −3, 75; p = 0.005, paired t-test; vehicle and FK506, respectively). However, FK506-treated animals exhibited enhanced OL memory when tested one week after training (t(15) = 2, 86; p = 0.01, unpaired t-test). Both groups did not differ in total exploration time (t(32) = 0, 91; p = 0.36; unpaired t-test; Fig. 5B), suggesting that FK506 did not affect exploratory activity.

Figure 5: Calcineurin activation induces forgetting of the OL memory. (A) Compared to saline-treated group, rats treated with FK506, an inhibitor of the protein phosphatase calcineurin, significantly spent more time exploring the new object location during the drug-free retention test conducted 7 d after learning. White bars represent the performance in the training, and blue bars in the test. (B) The overall exploratory activity indicates no differences among groups. Data expressed as mean ± SEM. *p < 0.5, **p < 0.01 (n = 8–9 per group). Full size image

This outcome confirms that calcineurin plays an essential role in forgetting of long-term memory.