A Novel Retrieval-Dependent Memory Process Revealed by the Arrest of ERK1/2 Activation in the Basolateral Amygdala

Abstract

Fully consolidated fear memories can be maintained or inhibited by retrieval-dependent mechanisms depending on the degree of re-exposure to fear cues. Short exposures promote memory maintenance through reconsolidation, and long exposures promote inhibition through extinction. Little is known about the neural mechanisms by which increasing cue exposure overrides reconsolidation and instead triggers extinction. Using auditory fear conditioning in male rats, we analyzed the role of a molecular mechanism common to reconsolidation and extinction of fear, ERK1/2 activation within the basolateral amygdala (BLA), after intermediate conditioned stimulus (CS) exposure events. We show that an intermediate re-exposure (four CS presentations) failed to activate ERK1/2 in the BLA, suggesting the absence of reconsolidation or extinction mechanisms. Supporting this hypothesis, pharmacologically inhibiting the BLA ERK1/2-dependent signaling pathway in conjunction with four CS presentations had no effect on fear expression, and the NMDA receptor partial agonist d-cycloserine, which enhanced extinction and ERK1/2 activation in partial extinction protocols (seven CSs), had no behavioral or molecular effect when given in association with four CS presentations. These molecular and behavioral data reveal a novel retrieval-dependent memory phase occurring along the transition between conditioned fear maintenance and inhibition. CS-dependent molecular events in the BLA may arrest reconsolidation intracellular signaling mechanism in an extinction-independent manner. These findings are critical for understanding the molecular underpinnings of fear memory persistence after retrieval both in health and disease.SIGNIFICANCE STATEMENT Consolidated fear memories can be altered by retrieval-dependent mechanisms. Whereas a brief conditioned stimulus (CS) exposure promotes fear memory maintenance through reconsolidation, a prolonged exposure engages extinction and fear inhibition. The nature of this transition and whether an intermediate degree of CS exposure engages reconsolidation or extinction is unknown. We show that an intermediate cue exposure session (four CSs) produces the arrest of ERK1/2 activation in the basolateral amygdala, a common mechanism for reconsolidation and extinction. Amnestic or hypermnestic treatments given in association with four CSs had no behavioral or molecular effects, respectively. This evidence reveals a novel retrieval-dependent memory phase. Intermediate degrees of CS exposure fail to trigger reconsolidation or extinction, leaving the original memory in an insensitive state.

Keywords: ERK1/2; NMDAR; extinction; limbo; reconsolidation.

Figure 1.

Intermediate cue exposure fails to activate BLA ERK1/2, a molecular marker of fear memory reconsolidation and extinction. A, Experimental design. Rats were fear conditioned with two CS–US pairings. Twenty-four hours after training, animals were divided into the following five groups: NR control, and 1, 4, 7, and 10 CS presentations (1CS, 4CS, 7CS, or 10CS, respectively). Twenty minutes after the first CS presentation, or straight from the home cage, animals were killed and BLA cytosolic protein extracts prepared. B, Mean (±SEM) percentage of time spent freezing during cued fear conditioning and at different number of cue exposure sessions (NR, n = 23; 1CS, n = 12; 4CS, n = 10; 7CS, n = 8; 10CS, n = 12). C, Representative Western blot picture and analysis of pERK1/2 levels in the BLA for NR, 1CS, 4CS, 7CS, and 10CS groups. Mean relative optical density as the percentage of NR (±SEM) shows that pERK1/2 is increased after 1 or 10 CS presentations, but is unchanged after 4 or 7CSs. TR, Training session. *p < 0.05.

Figure 2.

Injector tip placements within the basolateral amygdala for each experimental condition: 1, 4, 7, and 10 CSs. Vehicle: open circles; U0126: closed circles. This figure was modified, with permission, from Paxinos and Watson (1998).

Figure 3.

Intra-BLA administration of U0126, a specific inhibitor of ERK1/2 pathway, has no behavioral consequences during intermediate CS exposure. A, Experimental design. Animals were trained with two CS–US pairings. Twenty-four hours later, they were injected with vehicle or U0126 (1 μg/side) and within each drug condition divided into four groups (1CS, 4CS, 7CS, and 10CS) depending on the number of cue presentations. Twenty-four hours later, all the animals were tested for fear memory with the presentation of one CS (1CS VEH and 4CS U0126, n = 11; 10CS VEH, n = 7; remaining groups; n = 10 per group). B, C, Mean percentage of time spent freezing (±SEM) at CS exposure sessions (B) or long-term memory test (C) are shown. TS, Test session. *p < 0.05; **p < 0.01.

Figure 4.

NMDAR-dependent BLA ERK1/2 activation distinguishes between sensitive and insensitive transitional states between fear memory reconsolidation and extinction. A, Experimental design. Twenty-four hours after training, animals were intraperitoneally injected with saline or MK-801 (0.1 mg/kg) and then exposed to one CS (1CS groups) or returned to the home cage (NR groups). Fifty minutes after the injection, the animals were killed and BLA cytosolic protein extracts prepared. Representative Western blot pictures. Bar graph shows the mean BLA level of pERK1/2 (±SEM) as a percentage of NR group (n = 8 per group). Open bars, saline injection; striped bars, MK-801 injection. B, Experimental design and bar graph: same as for A, but after intraperitoneal injection the animals were either returned to the home cage (NR groups) or exposed to 10 CS presentations (10CS groups, n = 8 per group). C, Experimental design. Twenty-four hours after training, animals were intraperitoneally injected with saline or DCS (15 mg/kg) and then exposed to four CSs (4CS groups) or returned to the home cage (NR groups). Fifty minutes after the injection, the animals were killed and BLA cytosolic protein extracts prepared. Representative Western blot pictures. Bar graph shows the mean BLA level of pERK1/2 (±SEM) as a percentage of NR group (n = 5 per group). Open bars, saline injection; striped bars, DCS injection. D, Experimental design and bar graph: same as for C, but after intraperitoneal injection the animals were either returned to the home cage (NR groups) or exposed to seven CS presentations (7CS groups, n = 5 per group). *p < 0.05 vs NR group receiving the same intraperitoneal injection.