Memory Destabilization and Reconsolidation Dynamically Regulate the PKMζ Maintenance Mechanism

Abstract

Useful memory must balance between stability and malleability. This puts effective memory storage at odds with plasticity processes, such as reconsolidation. What becomes of memory maintenance processes during synaptic plasticity is unknown. Here we examined the fate of the memory maintenance protein PKMζ during memory destabilization and reconsolidation in male rats. We found that NMDAR activation and proteasome activity induced a transient reduction in PKMζ protein following retrieval. During reconsolidation, new PKMζ was synthesized to re-store the memory. Failure to synthesize new PKMζ during reconsolidation impaired memory but uninterrupted PKMζ translation was not necessary for maintenance itself. Finally, NMDAR activation was necessary to render memories vulnerable to the amnesic effect of PKMζ-antisense. These findings outline a transient disruption and renewal of the PKMζ memory maintenance mechanism during plasticity. We argue that dynamic changes in PKMζ protein levels can serve as an exemplary model of the molecular changes underlying memory destabilization and reconsolidation.SIGNIFICANCE STATEMENT Maintenance of long-term memory relies on the persistent activity of PKMζ. However, after retrieval, memories can become transiently destabilized and must be reconsolidated within a few hours to persist. During this period of plasticity, what happens to maintenance processes, such as those involving PKMζ, is unknown. Here we describe dynamic changes to PKMζ expression during both destabilization and reconsolidation of auditory fear memory in the amygdala. We show that destabilization induces a NMDAR- and proteasome-dependent loss of synaptic PKMζ and that reconsolidation requires synthesis of new PKMζ. This work provides clear evidence that memory destabilization disrupts ongoing synaptic maintenance processes which are restored during reconsolidation.

Keywords: PKMζ; amygdala; destabilization; maintenance; memory; reconsolidation.

Figure 1.

Synaptic PKMζ expression decreases within 1 h after retrieval of an auditory fear conditioning memory. A, For those rats that were re-exposed to the tone conditioned stimulus (CS), freezing behavior was considerably elevated during the tone (CS) than to the context, before the onset of the tone (Pre-CS, paired-samples t test, t₍₅₎ = −7.222, p = 0.001). B, Rats trained in auditory fear conditioning and killed 1 h after retrieval showed significantly lower PKMζ protein in BLA synapses compared with rats that did not undergo retrieval (independent-samples t test, t₍₉₎ = 2.755, p = 0.022). C, Rats were trained with tone and no shock. For those rats that were re-exposed to the tone, freezing behavior was equally low to both the context and the tone (paired-samples t test, t₍₇₎ = −1.409, p = 0.202). D, Without prior tone-shock pairing, rats re-exposed to the tone showed similar PKMζ protein in BLA tissue compared with rats that were not re-exposed to the tone (independent-samples t test, t₍₁₄₎ = 0.378, p = 0.711). Data are mean ± SEM. *p < 0.05. ***p < 0.001.

Figure 2.

Post-retrieval reduction of PKMζ is destabilization-dependent. A, Freezing behavior at retrieval test. Both APV- and vehicle-infused rats showed similar freezing behavior (independent-samples t test, t₍₁₈₎ = 0.746, p = 0.465). B, PKMζ protein levels from Western blots. Rats that received APV infusion before retrieval showed significantly higher PKMζ protein compared with rats that received vehicle (independent-samples t test, t₍₁₈₎ = 2.358, p = 0.030). C, PKMζ protein levels from Western blots. Rats that received APV infusion without retrieval showed similar PKMζ protein expression compared with vehicle-infused rats (independent-samples t test, t₍₁₅₎ = 0.098, p = 0.923). D, Freezing behavior at retrieval test. Both β-lac- and vehicle-infused rats showed similar freezing behavior (independent-samples t test, t₍₁₂₎ = 0.755, p = 0.465). E, PKMζ protein levels from Western blots. Rats that received β-lac infusion after retrieval showed significantly higher PKMζ protein compared with rats that received vehicle (independent-samples t test, t₍₁₂₎ = 2.377, p = 0.035). F, PKMζ protein levels from Western blots. Rats that received β-lac infusion without retrieval showed similar PKMζ protein expression compared with vehicle-infused rats (independent-samples t test, t₍₁₂₎ = −0.169, p = 0.869). Data are mean ± SEM. *p < 0.05.

Figure 3.

Increased expression of PKMζ following memory reconsolidation. A, Freezing behavior from rats killed either 1 or 24 h after retrieval. Both groups showed similar freezing to the tone at the retrieval test (independent-samples t test, t₍₁₃₎ = −0.064, p = 0.950). B, PKMζ protein levels from Western blots. Rats killed 1 h after retrieval showed significantly lower PKMζ protein in BLA tissue compared with rats killed 24 h after retrieval (independent-samples t test, t₍₁₃₎ = 2.365, p = 0.034). Data are mean ± SEM. *p <0.05.

Figure 4.

PKMζ-antisense disrupts memory reconsolidation. A, Behavior data showed a significant effect of the infusion on performance (one-way repeated-measures ANOVA, F_(1,18) = 11.005, p = 0.004). Rats that received PKMζ-antisense showed a significant impairment at the PRLTM test 24 h after infusion (Tukey's test, p < 0.001), whereas rats that received the scrambled sequence showed no impairment (Tukey's test, p = 0.210). B, Behavior data showed no effect of infusion on performance when the infusion occurred 24 h after retrieval (one-way repeated-measures ANOVA, F_(1,14) = 0.055, p = 0.818). Data are mean ± SEM. ***p < 0.001.

Figure 5.

NMDAR activation renders memory vulnerable to impairment by PKMζ-antisense. Behavior data revealed an interaction of pre-retrieval drug and post-retrieval infusion on performance (two-way repeated-measures ANOVA, F_(1,31) = 6.65, p = 0.015). That is, PKMζ-antisense had an amnesic effect in vehicle-treated animals (Tukey's test, t₍₃₁₎ = 5.045, p < 0.001) but not in APV-treated rats (Tukey's test, t₍₃₁₎ = −0.5428, p = 0.999). Rats receiving the scrambled ODN sequence showed no impairment in performance. Data are mean ± SEM. ***p < 0.001.