Role of circadian rhythm and REM sleep for memory consolidation

Abstract

Although sleep is strongly implicated in memory consolidation, the molecular basis for the role of sleep in memory is not known. It has been established that the consolidation of hippocampus-dependent memory depends on the activation of the Erk1,2 MAP kinase (MAPK) pathway which activates de novo CRE-mediated transcription and translation, two processes required for memory consolidation pathway. The activation of MAPK during memory formation and its nuclear translocation both depend upon cAMP signals generated by the calmodulin-stimulated adenylyl cyclases, type 1 and type 8 (AC1 and AC8). This signaling pathway undergoes a circadian oscillation in the hippocampus with maximal activation during REM sleep. This data supports the hypothesis that the persistence of long-term memory traces may depend upon the reactivation and circadian oscillation of the cAMP/MAP kinase/CRE transcriptional pathway in tagged neurons which reaches a maximum during REM sleep.

Keywords: Adenylyl cyclase; CREB; Calcium; Circadian; MAP kinase; Memory; REM Sleep; cAMP.

Fig. 1.

Hippocampus memory formation depends on MAPK stimulation. MAPK activation is required for consolidation of hippocampus-dependent memory because it mediates Ca²⁺ stimulation of protein synthesis and CRE-mediated transcription, two events required for memory consolidation.

Fig. 2.

Stimulation of CRE-mediated transcription when mice are trained for contextual memory. a. Summary of associative learning measured eight hours after training. Mean percentage of time spent freezing in the conditioning chamber is depicted for naive, unpaired control and context-trained mice (naive, n = 7; unpaired, n = 8; context-trained, n = 23; naive versus context and unpaired versus context, p < 0.0005). (b) Low-magnification confocal images showing CRE-regulated Lac Z immunostaining in hippocampal slices from representative naive, unpaired control and context-trained mice. Scale bar represents 500 m (c) Higher-magnification images of the CA1 region from representative naive, unpaired control and context-trained mice. Scale bar represents 100 microns (Impey et al. 1998c).

Fig. 3.

Activation of MAPK in area CA1 following training for context. Mice were trained for context and sacrificed at various times after shocking. (a) MAPK activity in the hippocampus was monitored by Western analysis for p-44 and p-42. C is the unpaired control. Western blots were scanned and the data was normalized to unphosphorylated MAPK scanned on the same blot. (b) Hippocampal sections were stained for p-MAPK (red) and Hoechst (blue). Unpaired controls were shocked immediately when placed in context and do not develop contextual memory (Sindreu et al. 2007).

Fig. 4.. Translation pathways regulating protein synthesis are activated after contextual fear conditioning.

A, Western analysis of CA1 lysates from wild type mice 30 min. after training for contextual fear. Mice showed increase in phosphorylation of T187/Y189 of Erk2 (p-Erk2), S209 phosphorylation of eIF4E (p-eIF4E), S235/236 phosphorylation of S6 (p-S6), and T36/45 phosphorylation of 4EBP1 (p-4EBP1) compared to control (shock). B and C, quantification of changes observed in phosphorylated proteins. Data is Mean ± Standard error of mean (sem), and is expressed as a percentage of control (shock). N (number of mice) is 5 each for control and context- shock vs. control (shock) using student’s t-test (Saraf et al. 2014).

Fig. 5.. Translation initiation shows a diurnal oscillation in the hippocampus.

Mice maintained on 12-h light /12-h dark cycle were sacrificed every 4 hr. A, hippocampal lysates obtained from wild type mice during the 24 hr (ZT0 to ZT20 hr) were evaluated by Western analysis for the phosphorylated Ser209 eIF4E and total eIF4E levels. p-eIF4E/eIF4E levels were elevated at ZT4 hr (*p< 0.05 vs. ZT0 hr, One-Way ANOVA followed by Dunnett’s post hoc test) and ZT8 hr (***p<0.001 vs. ZT0 hr, One-Way ANOVA followed by Dunnett’s post hoc test). B, hippocampal lysates from wild type mice were analyzed by Western analysis for the phosphorylated Thr36/45 4EBP1 and Erk2 (loading control) levels. p-4EBP1/Erk2 levels were increased at ZT4 h (*p< 0.05 vs. ZT0 h, One-Way ANOVA followed by Dunnett’s post hoc test) and ZT8 h (**p<0.01 vs. ZT0 h, One-Way ANOVA followed by Dunnett’s post hoc test). Data is shown as mean ± sem. C, Western analysis of hippocampal lysates from ZT4 hr and ZT16 hr for phosphorylated eIF4E, total eIF4E, phosphorylated 4EBP1 and actin (as a loading control). D, quantification of changes observed in phosphorylated eIF4E and 4EBP1 proteins normalized to total eIF4E and actin levels respectively, and expressed as a percentage of ZT 4hr. Data is shown as mean ± sem, ***p<0.001 ZT4 hr vs. ZT16 hr using student’s t-test (Saraf et al. 2014).

Fig. 6.. Administration of anisomycin to area CA1 of the hippocampus at ZT4–8 two days after training impairs the persistence of contextual memory.

A, Mice trained in contextual fear conditioning were infused daily for 4 days with vehicle or 50 μg anisomycin/hippocampus (between ZT4-ZT8 ), starting 48 h after training. Mice were tested for fear memory at day 14 and 28 after training. B, Anisomycin injected mice (N=6 animals) had reduced freezing during testing at both day 14 and 28 as compared with the vehicle group (N=9 animals). B, Anisomycin and vehicle injected mice were trained and tested (24 h later) for Step-through latency in passive avoidance. Data is expressed as mean ± sem, ***p<0.001, **p<0.01 vehicle vs. anisomycin group using student’s t-test (Saraf et al. 2014).

Fig. 7.. Mechanisms for initiation of translation through activation of CaM-stimulated adenylyl cyclases and MAPK.

It is hypothesized that Ca²⁺ signals generated during training for contextual memory activate calmodulin-stimulated adenylyl cyclases, which generate a cAMP signal for the activation and of MAPK. Stimulation of MAPK stimulates translation by several mechanisms, which lead to the phosphorylation and activation of eIF4E and S6.

Fig. 8.. Hypothesis for the persistence of hippocampus-dependent memory.

It is hypothesized that MAPK is activated during training because of upstream cAMP stimulation of Ras and/or Rap1 coupled with PKA inhibition of STEP phosphatase. The eventual decrease in pMAPK may be due to calcineurin reactivation of STEP. It is proposed that the persistence of hippocampus-dependent memory is dependent on the CaM-stimulated adenylyl cyclases and that memory is maintained by a circadian oscillation of this pathway in the hippocampus. It is hypothesized that the cAMP/MAPK/CREB transcriptional pathway reaches a maximum during the light phase of the circadian cycle, specifically in REM sleep.