Fluctuating NMDA Receptor Subunit Levels in Perirhinal Cortex Relate to Their Dynamic Roles in Object Memory Destabilization and Reconsolidation

Abstract

Reminder cues can destabilize consolidated memories, rendering them modifiable before they return to a stable state through the process of reconsolidation. Older and stronger memories resist this process and require the presentation of reminders along with salient novel information in order to destabilize. Previously, we demonstrated in rats that novelty-induced object memory destabilization requires acetylcholine (ACh) activity at M₁ muscarinic receptors. Other research predominantly has focused on glutamate, which modulates fear memory destabilization and reconsolidation through GluN2B- and GluN2A-containing NMDARs, respectively. In the current study, we demonstrate the same dissociable roles of GluN2B- and N2A-containing NMDARs in perirhinal cortex (PRh) for object memory destabilization and reconsolidation when boundary conditions are absent. However, neither GluN2 receptor subtype was required for novelty-induced destabilization of remote, resistant memories. Furthermore, GluN2B and GluN2A subunit proteins were upregulated selectively in PRh 24 h after learning, but returned to baseline by 48 h, suggesting that NMDARs, unlike muscarinic receptors, have only a temporary role in object memory destabilization. Indeed, activation of M₁ receptors in PRh at the time of reactivation effectively destabilized remote memories despite inhibition of GluN2B-containing NMDARs. These findings suggest that cholinergic activity at M₁ receptors overrides boundary conditions to destabilize resistant memories when other established mechanisms are insufficient.

Keywords: acetylcholine; boundary conditions; destabilization; glutamate; memory; reconsolidation.

Figure 1

Cannula placements in perirhinal cortex (PRh): (a) schematic of infusion tip placements from all rats used in behavioral experiments; (b) micrograph showing guide cannula tract with the infusion tip terminating near the rhinal sulcus.

Figure 2

GluN2B-containing NMDARs in PRh are involved in destabilizing, but not reconsolidating, relatively recent object memories. (a) Standard SOR parameters, with immediate pre-reactivation PRh infusions to target destabilization and prevent the impairing effects of post-reactivation infusions of the reconsolidation inhibitor anisomycin. (b) Standard SOR parameters, with only post-reactivation intra-PRh infusions to target memory reconsolidation. (c) Choice-phase DRs. All groups’ choice DRs differed significantly from their respective sample DR, with the exception of the veh–aniso group, suggesting memory was impaired in this group. The veh–veh and Ro–aniso groups had significantly better memory performance during the choice phase compared to the veh–aniso group. (d) Choice-phase DRs. Both groups’ choice DRs differed significantly from their respective sample DRs, suggesting memory was intact, while performance during the choice phase did not differ between groups. Bars are mean DR ± standard error of the mean (SEM); ** p < 0.01.

Figure 3

GluN2A-containing NMDARs in PRh are involved in reconsolidating, but not destabilizing, relatively recent object memories. (a) Standard SOR parameters, with intra-PRh infusions performed immediately prior to reactivation to target destabilization and prevent the impairing effects of post-reactivation infusion of anisomycin. (b) Standard SOR parameters, where only post-reactivation intra-PRh infusions were performed to target reconsolidation. (c) Choice-phase DRs. Only the veh–veh group had a choice DR that differed from its respective sample DR, suggesting this was the only group with intact memory. This group had significantly greater performance than all other groups. (d) Choice-phase DRs. The NVP group choice DR did not differ from its respective sample DR, suggesting lack of recognition memory, while memory was significantly impaired compared to the veh group. Bars are mean DR ± SEM; ** p < 0.01, *** p < 0.001.

Figure 4

GluN2B-containing NMDARs in PRh are not involved in destabilizing or reconsolidating remote (48h) object memories. (a) Remote SOR parameters, where contextual novelty in the form of a floor insert is required at the time of reactivation to destabilize remote object memories. PRh infusions were performed immediately prior to reactivation to target destabilization and prevent the impairing effects of post-reactivation infusion of anisomycin. (b) Remote SOR parameters, where only post-reactivation PRh infusions were performed to target reconsolidation. (c) Choice-phase DRs. The veh–veh and Ro–veh groups had choice DRs that differed from their respective sample DRs, suggesting that these groups had intact memory. Memory in both groups receiving post-reactivation anisomycin was significantly impaired compared to vehicle conditions. (d) Choice-phase DRs, demonstrating no differences in memory performance. Both groups’ choice DRs differed significantly from their respective sample DRs, suggesting memory was intact. Bars are mean DR ± SEM; * p < 0.05.

Figure 5

GluN2A-containing NMDARs in PRh are not required for destabilizing or reconsolidating remote object memories. (a) Remote SOR parameters, where a floor insert is required at the time of reactivation to destabilize remote object memories. PRh infusions were performed immediately prior to reactivation to target destabilization and prevent the impairing effects of post-reactivation infusion of anisomycin. (b) Remote SOR parameters, where only post-reactivation PRh infusions were performed to target reconsolidation. (c) Choice-phase DRs demonstrating that both groups receiving post-reactivation anisomycin were significantly impaired compared to the veh–veh condition. The veh–veh and NVP–veh groups had choice DRs that differed from their respective sample DRs, suggesting that only these groups had intact memory. (d) Choice-phase DRs demonstrating memory did not differ between groups. Both groups’ choice DRs differed significantly from their respective sample DRs, suggesting memory was intact. Bars are mean DR ± SEM; * p < 0.05, *** p < 0.001.

Figure 6

Destabilization of remote memories promoted by M₁-receptor activation in PRh does not require GluN2B-containing NMDARs. (a) Remote SOR parameters were used, but the contextual novelty was withheld during reactivation and instead destabilization was induced by activating M₁-receptors in PRh prior to reactivation. (b) Choice-phase DRs. Groups that received anisomycin following reactivation had significantly impaired memory performance compared to post-reactivation veh conditions, suggesting that CDD infusion prior to reactivation was sufficient to induce destabilization and that co-infusion of Ro-25 did not prevent M₁-induced destabilization. Bars are mean DR ± SEM; ** p < 0.01, *** p < 0.001.

Figure 7

GluN2A, GluN2B, and GluN1 levels are significantly greater 24 h following learning compared to 48 h in PRh but not HPC. (a) Schematic of behavioral parameters. Rats in the learning group underwent a series of sample phases, then PRh and HPC tissues were collected either 24 or 48 h later. Non-learning controls remained in home cage. (b) Representative blot of GluN2A in anterior (A) and posterior (P) PRh in learning (+) and non-learning (-) groups 24 and 48 h following learning. GluN2A in whole PRh was significantly greater 24 h following learning compared to non-learning controls and 48 h following learning. (c) Representative blot of GluN2B in anterior and posterior PRh. GluN2B was significantly increased in whole PRh 24 h following learning compared to non-learning controls and 48 h following learning. (d) Representative blot of GluN1 in anterior and posterior PRh. GluN1 in PRh was significantly greater 24 vs 48 h following learning, but was not different from non-learning controls. (e) Representative blot of GluN2A in HPC. GluN2A levels did not differ across groups at 24 or 48 h following learning. (f) Representative blot of GluN2B in HPC. GluN2B levels did not differ across groups at 24 or 48 h following learning. (g) Representative blot of GluN1 in HPC. GluN1 levels did not differ across groups 24 or 48 h following learning. Bars represent mean GluN2 or GluN1 target–actin normalized to control ± SEM; * p < 0.05, ** p < 0.01.