Active Transition of Fear Memory Phase from Reconsolidation to Extinction through ERK-Mediated Prevention of Reconsolidation

Abstract

The retrieval of fear memory induces two opposite memory process, i.e., reconsolidation and extinction. Brief retrieval induces reconsolidation to maintain or enhance fear memory, while prolonged retrieval extinguishes this memory. Although the mechanisms of reconsolidation and extinction have been investigated, it remains unknown how fear memory phases are switched from reconsolidation to extinction during memory retrieval. Here, we show that an extracellular signal-regulated kinase (ERK)-dependent memory transition process after retrieval regulates the switch of memory phases from reconsolidation to extinction by preventing induction of reconsolidation in an inhibitory avoidance (IA) task in male mice. First, the transition memory phase, which cancels the induction of reconsolidation, but is insufficient for the acquisition of extinction, was identified after reconsolidation, but before extinction phases. Second, the reconsolidation, transition, and extinction phases after memory retrieval showed distinct molecular and cellular signatures through cAMP responsive element binding protein (CREB) and ERK phosphorylation in the amygdala, hippocampus, and medial prefrontal cortex (mPFC). The reconsolidation phase showed increased CREB phosphorylation, while the extinction phase displayed several neural populations with various combinations of CREB and/or ERK phosphorylation, in these brain regions. Interestingly, the three memory phases, including the transition phase, showed transient ERK activation immediately after retrieval. Most importantly, the blockade of ERK in the amygdala, hippocampus, or mPFC at the transition memory phase disinhibited reconsolidation-induced enhancement of IA memory. These observations suggest that the ERK-signaling pathway actively regulates the transition of memory phase from reconsolidation to extinction and this process functions as a switch that cancels reconsolidation of fear memory.SIGNIFICANCE STATEMENT Retrieval of fear memory induces two opposite memory process; reconsolidation and extinction. Reconsolidation maintains/enhances fear memory, while extinction weakens fear memory. It remains unknown how memory phases are switched from reconsolidation to extinction during retrieval. Here, we identified an active memory transition process functioning as a switch that inhibits reconsolidation. This memory transition phase showed a transient increase of extracellular signal-regulated kinase (ERK) phosphorylation in the amygdala, hippocampus and medial prefrontal cortex (mPFC). Interestingly, inhibition of ERK in these regions at the transition phase disinhibited the reconsolidation-mediated enhancement of inhibitory avoidance (IA) memory. These findings suggest that the transition memory process actively regulates the switch of fear memory phases of fear memory by preventing induction of reconsolidation through the activation of the ERK-signaling pathway.

Keywords: ERK; extinction; fear memory; reconsolidation; transition.

Figure 1.

Memory phases after retrieval in the IA task. A, Experimental design. B, Re-exposure to the light compartment until the mouse entered the dark compartment. The VEH group showed enhancement of IA memory (n = 7). The ANI group showed disruption of reactivated IA memory (n = 7). C, D, Re-exposure to the dark compartment for 3 min (C) or 10 min (D) following re-exposure to the light compartment. The VEH groups showed the long-term extinction of IA memory, while ANI blocked this (C, VEH, n = 8, ANI, n = 8; D, VEH, n = 10, ANI, n = 10). E, Re-exposure to the dark compartment for 1 min following re-exposure to the light compartment (VEH, n = 10, ANI, n = 10); *p < 0.05, **p < 0.01; post hoc Bonferroni's test; #p < 0.05, ##p < 0.01; paired t test. VEH, vehicle; ANI, anisomycin. PR-LTM, postreactivation long-term memory test. Error bars, SEM.

Figure 2.

Single (pCREB⁺/pERK^– neurons) or two (pCREB⁺/pERK^– and pCREB⁺/pERK⁺ neurons) populations of neurons appear in the amygdala (LA and BA regions) in the reconsolidation and extinction phases, respectively. A, Representative immunohistochemical staining of pCREB⁺, pERK⁺, and pCREB⁺/pERK⁺ cells in the amygdala (LA and BA regions) from the indicated groups. Scale bar: 100 μm. pCREB⁺ (B, G), pERK⁺ (C, H), pCREB⁺/pERK⁺ (D, I), pCREB⁺/pERK^– (E, J), and pCREB^–/pERK⁺ (F, K) cells in the LA (B–F) and BA (G–K) regions of the amygdala (NR, n = 6; Recon, n = 9; Tran, n = 9; Ext, n = 9); #p < 0.05, compared with the NR and Tran groups (B, E, G, J); ⁺p < 0.05, compared with the NR group (B, G, J); *p < 0.05, compared with the other groups (C, D, H, I). BA, basolateral region of the amygdala; LA, lateral region of the amygdala; NR, non-reactivated group; Recon, reconsolidation group; Tran, transition group; Ext, extinction group. Error bars, SEM.

Figure 3.

pCREB⁺/pERK^– neurons or pCREB^–/pERK⁺ neurons appear in the CA1 region of the hippocampus in the reconsolidation and extinction phases, respectively. A, Representative immunohistochemical staining of pCREB⁺, pERK⁺, and pCREB⁺/pERK⁺ cells in the CA1 region from the indicated groups. Scale bar: 100 μm. pCREB⁺ (B), pERK⁺ (C), pCREB⁺/pERK⁺ (D), pCREB⁺/pERK^– (E), and pCREB^–/pERK⁺ (F) cells in the CA1 region of the hippocampus (NR, n = 6; Recon, n = 9; Tran, n = 9; Ext, n = 9); #p < 0.05, compared with the NR and Recon groups (C, F); *p < 0.05, compared with the other groups (B, E). NR, non-reactivated group; Recon, reconsolidation group; Tran, transition group; Ext, extinction group. Error bars, SEM

Figure 4.

Single (pCREB⁺/pERK^– neurons) or three (pCREB⁺/pERK^–, pCREB^–/pERK⁺, and pCREB⁺/pERK⁺ neurons) populations of neurons appear in the mPFC (PL and IL regions) in the reconsolidation and extinction phases, respectively. A, Representative immunohistochemical staining of pCREB⁺, pERK⁺, and pCREB⁺/pERK⁺ cells in the mPFC (PL and IL regions) from the indicated groups. Scale bar: 100 μm. pCREB⁺ (B, G), pERK⁺ (C, H), pCREB⁺/pERK⁺ (D, I), pCREB⁺/pERK^– (E, J), and pCREB^–/pERK⁺ (F, K) cells in the PL (B–F) and IL (G–K) regions of the mPFC (NR, n = 6; Recon, n = 9; Tran, n = 9; Ext, n = 9); #p < 0.05, compared with the NR and Tran groups (B, E, G, J); ⁺p < 0.05, compared with the NR group (I); *p < 0.05, compared with the other groups (C, D, F, H, I, K). IL, infralimbic region of the mPFC; PL, prelimbic region of the mPFC; NR, non-reactivated group; Recon, reconsolidation group; Tran, transition group; Ext, extinction group. Error bars, SEM.

Figure 5.

Time course analysis of the phosphorylation levels of CREB and ERK following the reactivation session. A, Experimental design. B–D, Representative immunohistochemical staining of pCREB⁺ and pERK⁺ cells in the amygdala (B), CA1 region of the hippocampus (C), and mPFC (D) of the indicated groups. Scale bar: 100 μm. E, pCREB levels in the amygdala, mPFC, and CA1 region of the hippocampus after reactivation (n = 6–7 for each group). F, pERK levels in the amygdala, mPFC, and CA1 region of the hippocampus after reactivation (n = 6–7 for each group). The dashed line represents the NR group; *p < 0.05, compared with the NR group; +p < 0.05, compared with the other groups at 30 min after the reactivation session; post hoc Newman–Keuls test; #p < 0.05; unpaired t test. LA, lateral region of the amygdala; BA, basolateral region of the amygdala; PL, prelimbic region of the mPFC; IL, infralimbic region of the mPFC; NR, non-reactivated group; Recon, reconsolidation group; Tran, transition group; Ext, extinction group. Error bars, SEM.

Figure 6.

Inhibition of ERK in the mPFC, amygdala, or hippocampus blocks the reconsolidation/enhancement and long-term extinction of IA memory. A, B, Effects of U0126 microinfusion into the mPFC immediately (A) or at 30 min (B) after the reactivation session (re-exposure to the dark compartment for 0 min; A, VEH, n = 10, U0126, n = 11; B, VEH, n = 10, U0126, n = 10). C, D, Effects of U0126 microinfusion into the mPFC immediately (C) or at 30 min (D) after the reactivation session (re-exposure to the dark compartment for 10 min; C, VEH, n = 10, U0126, n = 10; D, VEH, n = 10, U0126, n = 10). E, F, Effects of U0126 microinfusion into the amygdala (E) or hippocampus (F) immediately after the reactivation session (re-exposure to the dark compartment for 0 min; E, VEH, n = 10, U0126, n = 10; F, VEH, n = 10, U0126, n = 10). G, H, Effects of U0126 microinfusion into the amygdala (G) or hippocampus (H) immediately after the reactivation session (re-exposure to the dark compartment for 10 min; G, VEH, n = 9, U0126, n = 9; H, VEH, n = 10, U0126, n = 10); *p < 0.05, **p < 0.01; post hoc Bonferroni's test; #p < 0.05, ##p < 0.01; paired t test. AMY, amygdala; HP, hippocampus; VEH, vehicle; DC, dark compartment. PR-LTM, postreactivation long-term memory test. Error bars, SEM.

Figure 7.

Inhibition of ERK in the mPFC, amygdala, or hippocampus disinhibits reconsolidation/enhancement of IA memory. A–C, Effects of U0126 microinfusion into the mPFC (A), AMY (B), or HP (C) immediately after the reactivation session (re-exposure to the dark compartment for 1 min; A, VEH, n = 12, U0126, n = 11; B, VEH, n = 10, U0126, n = 11; C, VEH, n = 10, U0126, n = 12). D–F, Systemic injection of a high dose of SL327 (20 mg/kg) blocked the transition (D), reconsolidation (E), and extinction (F) of IA memory (D, VEH, n = 10, SL327 10 mg/kg, n = 10, SL327 20 mg/kg, n = 11; E, VEH, n = 10, SL327 20 mg/kg, n = 10; F, VEH, n = 10, SL327 20 mg/kg, n = 10); *p < 0.05, **p < 0.01; post hoc Bonferroni's test; #p < 0.05, ##p < 0.01; paired t test. AMY, amygdala; HP, hippocampus; VEH, vehicle; SL 10, SL327 10 mg/kg; SL 20, SL327 20 mg/kg; DC, dark compartment. PR-LTM, postreactivation long-term memory test. Error bars, SEM.

Figure 8.

Cannula tip placement in the amygdala, hippocampus, and mPFC. A–K, Cannula tip placement from mice infused with each drug shown in Figures 6A (A), B (B), C (C), D (D), E (E), F (F), G (G), H (H) and 7A (I), B (J), C (K). Schematic drawing of coronal sections from all microinfused animals (amygdala, 1.34 mm posterior to bregma; hippocampus, 1.94 mm posterior to bregma; mPFC, 1.94 mm anterior to bregma). Only mice with needle tips within the boundaries of the amygdala, hippocampus, or mPFC were included in the data analysis. VEH: vehicle.