ERK/MAPK regulates hippocampal histone phosphorylation following contextual fear conditioning

Abstract

Long-term memory formation is regulated by many distinct molecular mechanisms that control gene expression. An emerging model for effecting a stable, coordinated pattern of gene transcription involves epigenetic tagging through modifications of histones or DNA. In this study, we investigated the regulation of histone phosphorylation in the hippocampus by the ERK/MAPK (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathway. We found that activation of ERK/MAPK in vitro significantly increased histone H3 phosphorylation in hippocampal area CA1. Furthermore, we found that contextual fear conditioning in vivo leads to a rapid time-dependent increase in histone H3 phosphorylation in area CA1. This increase paralleled the time course of contextual fear-dependent activation of ERK, and was inhibited in vivo by a latent inhibition paradigm as well as by injection of an N-methyl-d-aspartic acid receptor (NMDA-R) antagonist. Finally, injection of an inhibitor of MEK (MAP kinase/ERK kinase), the unique dual-specificity kinase upstream of ERK, blocked the increase in histone H3 phosphorylation seen after contextual fear conditioning. These results demonstrate that changes in histone phosphorylation in the hippocampus are regulated by ERK/MAPK following a behavioral fear conditioning paradigm.

Figure 1.

Activation of ERK regulates histone H3 phosphorylation, acetylation, and phospho-acetylation in vitro. (A) Quantification of immunoblot densities for phospho-ERK and total ERK. Treatment of hippocampal slices with PDA (3 μM, n = 9) or FSK (50 μM with 100 μM Ro20-1724, n = 9) for 1 h significantly increased phosphorylation of ERK2 in area CA1. Pre-incubation of slices with U0126 (20 μM, n = 5) for 10 min followed by PDA or FSK blocked the increase in ERK2 phosphorylation. Total ERK protein was unchanged in all treatments. Representative immunoblots for P-ERK and total ERK are shown for each treatment condition. Control (C) samples appear on the left, and experimental (E) samples appear on the right. (B) Quantification of immunoblot densities for phospho-histone H3, acetyl-histone H3, and phospho-acetyl-histone H3. Treatment of hippocampal slices with PDA (3 μM, n = 7) or FSK (50 μM with 100 μM Ro20-1724, n = 6) for 1 h significantly increased phosphorylation, acetylation, and phospho-acetylation of histone H3 in area CA1. Pre-incubation of slices with U0126 (20 μM, n = 5) for 10 min followed by PDA or FSK blocked these changes. Total histone H3 protein was unchanged. Representative immunoblots for P-H3 and total H3 are shown for each treatment condition. Control (C) samples appear on the left, and experimental (E) samples appear on the right. All drug-treated slices were compared with vehicle-treated controls. Error bars indicate standard error of the mean. Asterisks denote significant differences (P < 0.05) as determined by Tukey’s multiple comparison test.

Figure 2.

Contextual fear conditioning leads to formation of long-term fear memory and regulates ERK phosphorylation in vivo. (A) Quantification of freezing behavior 24 h following the training period. Animals trained under the contextual fear conditioning paradigm (FC, n = 12) displayed significantly greater freezing than either naive animals (naive, n = 4) or animals trained under the latent inhibition paradigm (lat inh, n = 12). Naive animals had not been previously exposed to either the training chamber or shocks. Animals injected with MK801 (300 μg/kg, n = 4) displayed significantly less freezing than animals injected with saline (0.9% NaCl, 1.25 mL/kg, n = 5). (B) Quantification of immunoblot densities for phospho-ERK and total ERK at different time points following contextual fear conditioning. ERK2 phosphorylation in area CA1 was significantly increased at 1 h after training (FC 1 h, n = 11) before returning to baseline and remaining even after 24 h. Total ERK was unchanged. Representative immunoblots for P-ERK and total ERK are shown for each time point. Control (C) samples appear on the left and experimental (E) samples appear on the right. (C) Quantification of immunoblot densities for phospho-ERK and total ERK. The latent inhibition paradigm (lat inh, n = 6) significantly reduced ERK2 phosphorylation in area CA1 compared with fear conditioning (FC 1 h, n = 11). Injection of animals with MK801 (300 μg/kg) prior to fear conditioning (FC + MK801, n = 3) significantly reduced ERK2 phosphorylation in area CA1 compared with injection with saline (0.9% NaCl, 1.25 mL/kg) prior to fear conditioning (FC + saline, n = 3). Total ERK was unchanged. Representative immunoblots for P-ERK and total ERK are shown for each condition. Control (C) samples appear on the left and experimental (E) samples appear on the right. Error bars indicate standard error of the mean. Asterisks denote significant differences (P < 0.05) as determined by Tukey’s multiple comparison test.

Figure 3.

Contextual fear conditioning regulates histone H3 phosphorylation, acetylation, and phospho-acetylation in vivo. (A) Quantification of immunoblot densities for phospho-histone H3, acetyl-histone H3, and phospho-acetyl-histone H3 at different time points following contextual fear conditioning. Histone H3 phosphorylation, acetylation, and phospho-acetylation in area CA1 were significantly increased at 1 h after training (FC 1 h, n = 8) before returning to baseline and remaining even after 24 h. Total histone H3 was unchanged. Representative immunoblots for P-H3 and total H3 are shown for each time point. Control (C) samples appear on the left, and experimental (E) samples appear on the right. (B) Quantification of immunoblot densities for phospho-histone H3, acetyl-histone H3, and phospho-acetyl-histone H3. The latent inhibition paradigm (lat inh, n = 4) significantly reduced histone H3 phosphorylation, acetylation, and phospho-acetylation in area CA1 compared with fear conditioning (FC 1 h, n = 8). Injection of animals with MK801 (300 μg/kg) prior to fear conditioning (FC + MK801, n = 3) significantly reduced histone H3 phosphorylation, acetylation, and phospho-acetylation in area CA1 compared with injection with saline (0.9% NaCl, 1.25 mL/kg) prior to fear conditioning (FC + saline, n = 3). Total histone H3 was unchanged. Representative immunoblots for P-H3 and total H3 are shown for each condition. Control (C) samples appear on the left, and experimental (E) samples appear on the right. Error bars indicate standard error of the mean. Asterisks denote significant differences (P < 0.05) as determined by Tukey’s multiple comparison test.

Figure 4.

MEK signaling through ERK contributes to long-term fear memory and contextual fear conditioning-induced changes in histone H3. (A) Quantification of freezing behavior 24 h following the training period. Animals that were fear conditioned and injected with DMSO (2.99 mL/kg, n = 4) displayed significantly greater freezing than either naive animals (naive, n = 4) or animals that were fear-conditioned and injected with SL327 (100 mg/kg, n = 7). Naive animals had not been previously exposed to either the training chamber or shocks. (B) Quantification of immunoblot densities for phospho-ERK and total ERK. Injection of animals with SL327 (100 mg/kg) following fear conditioning (FC + SL327, n = 9) significantly reduced ERK2 phosphorylation in area CA1 compared with injection with DMSO (2.99 mL/kg) following fear conditioning (FC + DMSO, n = 3). Total ERK was unchanged. Representative immunoblots for P-ERK and total ERK are shown for each condition. Control (C) samples appear on the left and experimental (E) samples appear on the right. (C) Quantification of immunoblot densities for phospho-histone H3, acetyl-histone H3, and phospho-acetyl-histone H3. Injection of animals with SL327 (100 mg/kg) following fear conditioning (FC + SL327, n = 3) significantly reduced histone H3 phosphorylation, acetylation, and phospho-acetylation in area CA1 compared with injection with DMSO (2.99 mL/kg) following fear conditioning (FC + DMSO, n = 3). Total histone H3 was unchanged. Representative immunoblots for P-H3 and total H3 are shown for each condition. Control (C) samples appear on the left, and experimental (E) samples appear on the right. Error bars indicate standard error of the mean. Asterisks denote significant differences (P < 0.05) as determined by Tukey’s multiple comparison test.