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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2011 Aug 1;108(33):13806–13811. doi: 10.1073/pnas.1104383108

Long-lasting behavioral responses to stress involve a direct interaction of glucocorticoid receptors with ERK1/2–MSK1–Elk-1 signaling

María Gutièrrez-Mecinas 1, Alexandra F Trollope 1, Andrew Collins 1, Hazel Morfett 1, Shirley A Hesketh 1, Flavie Kersanté 1, Johannes M H M Reul 1,1
PMCID: PMC3158237  PMID: 21808001

Abstract

Stressful events are known to have a long-term impact on future behavioral stress responses. Previous studies suggested that both glucocorticoid hormones and glutamate acting via glucocorticoid receptors (GRs) and N-methyl d-aspartate (NMDA) receptors, respectively, are of critical importance for the consolidation of these long-lasting behavioral responses at the dentate gyrus, the gateway of the hippocampal formation. We found that an acute psychologically stressful event resulted in ERK1/2 phosphorylation (pERK1/2), which within 15 min led to the activation of the nuclear kinases MSK1 and Elk-1 in granule neurons of the dentate gyrus. Next, MSK1 and Elk-1 activation evoked serine-10 phosphorylation and lysine-14 acetylation in histone H3, resulting in the induction of the neuroplasticity-associated immediate-early genes c-Fos and Egr-1 in these neurons. The pERK1/2-mediated activation of MSK1 and Elk-1 required a rapid protein–protein interaction between pERK1/2 and activated GRs. This is a unique nongenomic mechanism of glucocorticoid hormone action in dentate gyrus granule neurons on long-lasting behavioral responses to stress involving direct cross-talk of GRs with ERK1/2–MSK1–Elk-1 signaling to the nucleus.

Keywords: corticosterone, chromatin, epigenetics, hippocampus, memory

Adrenal glucocorticoid hormones play an important role in the behavioral consequences of stress (1). Glucocorticoids secreted during a stressful event facilitate learning of adaptive behavioral responses and the consolidation of memories of the event (1, 2). Aberrant glucocorticoid secretion, as a result of chronic stress, is implicated in stress-related disorders such as major depression and anxiety (3–5).

It is still unclear how glucocorticoid hormones affect behavior at the molecular level. Glucocorticoid levels attained after stress influence cellular function by activating glucocorticoid receptors (GRs) (6). These receptors bind to their target sites in gene promoters, thereby changing gene expression (7). Activated GRs can also interact through protein–protein interactions with a broad range of intracellular signaling molecules including transcription factors and enzymes (7). Whether GRs directly interact with intracellular signaling pathways to influence stress-related behavior is unknown.

A signaling pathway involved in behavioral adaptation and memory formation is the extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) signaling pathway (8). This pathway is activated through N-methyl d-aspartate receptors (NMDA-Rs) and other membrane receptors and is involved in changes in neuronal structure and function (8). Hippocampal NMDA-R–mediated ERK MAPK signaling is involved in behavioral responses observed in Morris water maze learning, contextual fear conditioning, and the forced swim test (9–11). In vitro experiments suggest that ERK MAPK signaling activates nuclear histone modifying enzymes such as MSK1 (mitogen- and stress-activated kinase 1) (12, 13) and Elk-1 (ETS domain protein-1) (14). These enzymes evoke changes in the chromatin structure underlying gene transcription. Both kinases are substrates of ERK1/2 (12–14). Phosphorylated MSK1 (pMSK1) phosphorylates serine-10 in histone H3 tails in various gene promoters (e.g., c-fos) (12, 13). pElk-1 binds to the serum response element (SRE) in distinct gene promoters [including the c-fos and egr-1 (early growth response protein 1) promoters], after which it recruits the histone acetyl transferase (HAT) p300 (15). Thus, MSK1 and Elk-1 activation results in histone H3 phosphorylation and acetylation in gene promoters, thereby activating gene transcription.

Blockade of NMDA-Rs, inhibition of ERK1/2 activation, and gene deletion of MSK1 in vivo prevent histone H3 phosphorylation and acetylation and c-Fos induction in the hippocampus and impair behavioral responses in the Morris water maze, contextual fear conditioning, and forced swim test (11, 16). Furthermore, glucocorticoid hormones secreted as a result of the behavioral challenge are of great importance in Morris water maze learning and contextual fear conditioning (17, 18). Moreover, epigenetic, gene expression, and behavioral responses to forced swimming (FS) and novelty critically depend on GR-mediated glucocorticoid action in dentate gyrus granule neurons (19–21). Thus, stress-induced behavioral responses involve activation of ERK MAPK and GR-mediated signaling in dentate neurons that converge and result in specific histone H3 modifications [i.e., serine-10 (S10) phosphorylation and lysine-14 (K14) acetylation (H3S10p-K14ac)] and activation of gene transcription (22). How these signaling pathways converge is presently unknown. We postulated that glucocorticoids enhance molecular and behavioral responses through a direct interaction of GRs with ERK1/2 and its downstream partners, MSK1 and Elk-1, in dentate gyrus neurons.

We show that after an acute stressful challenge glucocorticoid hormones via GRs rapidly enhance the pERK1/2-mediated generation of pMSK1 and pElk-1, leading to the phosphorylation and acetylation of histone H3 and induction of c-Fos and Egr-1 specifically in dentate neurons. Furthermore, GRs enhance ERK MAPK signaling to the chromatin and behavior through a physical interaction with pERK1/2 and pMSK1.

Results

Long-Term Glucocorticoid Effects on a Stress-Related Behavioral Response.

Fig. 1A shows that when rats are subjected to an acute FS challenge (i.e., 15 min in 25 °C water) they display, upon reexposure to the challenge 24 h later, substantially more immobility behavior than during the initial test. Fig. 1B shows that this behavioral response is maintained not just for 24 h but actually for at least 4 wk. The consolidation of this long-lasting behavioral response requires an action of glucocorticoid hormones via GRs, but not mineralocorticoid receptors (MRs), in the dentate gyrus during the initial test (21). Accordingly, GR antagonist (e.g., RU486) injection before the initial test results in an impaired behavioral immobility response in a retest 24 h later but also if tested 4 wk later (Fig. 1 A and B).

Fig. 1.

Fig. 1.

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Effect of the GR antagonist RU486 on FS-induced behavioral responses (A and B) and molecular responses in dentate neurons (C–E) of rats. Rats were treated with RU486 or vehicle 30 min before the first swim test and either retested 24 h later (A) or 4 wk (4W) later (B). Immobility behavior was expressed as arbitrary units (mean ± SEM, n = 15–18 rats except 4W-RU486 group: n = 8 rats per group). *P < 0.05, compared with the respective first test treatment group (paired t test after repeated measures ANOVA); #P < 0.05, compared with the vehicle-treated group within the same (re)test (unpaired t test). (C–E) Rats were treated with vehicle or RU486 and after 30 min were forced to swim for 15 min. Brains were collected at 60 min (C and D) or 90 min (E) after start of stress. Baseline animals were injected with vehicle or RU486 and killed at 90 min (C and D) or 120 min (E) after injection. The number of immunostained neurons in 50-μm sections of the dentate gyrus was determined by immunohistochemistry using anti-H3S10p-K14ac (C), –c-Fos (D) and –Egr-1 (E) antibodies. The data are shown as group means ± SEM (n = 4–10 rats per group) of the average number of stained dentate gyrus neurons in a 50-μm section. Bonferroni-corrected test with contrasts after two-way ANOVA: *P < 0.05, effect of FS compared with the respective vehicle or drug treatment group; #P < 0.05, significant effect of RU486 compared with the respective baseline or FS group.

Fig. 1 C–E shows that during the consolidation phase that follows the initial FS challenge H3S10p-K14ac, c-Fos, and Egr-1 are generated in dentate neurons. These responses occurred selectively in mature granule neurons of the dorsal blade (Fig. S1). Pretreatment with RU486 blocked the FS-induced responses in H3S10p-K14ac c-Fos, and Egr-1 (Fig. 1 C–E).

Stress Evokes Phosphorylation of ERK1/2, MSK1, and Elk-1 in Dentate Gyrus Granule Neurons.

We investigated the activation of ERK1/2 and its downstream substrates MSK1 and Elk-1 by immunohistochemical analyses of pERK1/2, pMSK1, pElk-1, H3S10p-K14ac, c-Fos, and Egr-1 in dentate neurons after FS (Fig. 2). The immunoreactive proteins presented a sparse staining pattern in dentate neurons. pERK1/2 staining was found in both the nuclear and cytoplasmic compartments (Fig. 2A), whereas staining of pMSK1, pElk-1, H3S10p-K14ac, c-Fos, and Egr-1 was only nuclear (Fig. 2 B–F). Furthermore, the stress-induced signaling was specific through pERK1/2 and pMSK1 as neither p-p38MAPK (another MSK1 kinase) nor pRSK1/2 (an MSK-like kinase) (23) was found after stress. The phosphorylation of ERK1/2, MSK1, and Elk-1 was a rapid, transient phenomenon peaking at 15 min after start of the FS challenge (Fig. 2 A–C). Fig. 1 D and E and previous work (11) show that H3S10p-K14ac and c-Fos peak at 1–2 h and return to baseline at 4 h (11). The increased expression of pERK1/2, pMSK1, and pElk-1 after FS occurred only in mature dentate neurons of the dorsal blade (Fig. S2). Egr-1 levels were elevated at 15 min and 90 min after FS (Fig. 2F). The fast increase after stress (i.e., at 15 min) has been reported to be MAPK independent, whereas the second increase at 90 min is MAPK dependent (24). The rise in Egr-1 at 15 min was found in young and mature dentate neurons of both dorsal and ventral blades, whereas the increase at 90 min was only observed in mature dorsal blade neurons (Fig. S1).

Fig. 2.

Fig. 2.

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Time courses of stress-evoked changes in pERK1/2 (A), pMSK1 (B), pElk-1 (C), H3S10p-K14ac (D), c-Fos (E), and Egr-1 (F) in the rat dentate gyrus. Rats were forced to swim (15 min in 25 °C water) and perfused at 15 min (FS15), 60 min (FS60), or 90 min (FS90) after the start of stress. A separate group of rats was perfused under baseline conditions (Bs). The images show immunostained neurons in the granular cell layer of the dentate gyrus. The bar diagrams present group means ± SEM (n = 3–9 rats per group) of the average number of immunopositive neurons in a 50-μm section of the dentate gyrus. *P < 0.05, significantly different from Bs (post hoc tests with contrasts after ANOVA analysis).

Colocalization of pERK1/2, pMSK1, pElk-1, H3S10p-K14ac, c-Fos, and Egr-1 in Dentate Gyrus Granule Neurons.

To study glucocorticoid interaction with NMDA-ERK MAPK signaling, colocalization of pERK1/2, pMSK1, and pElk-1 in dentate neurons needed to be demonstrated first. Dentate neurons express GRs and NMDA-Rs (6, 25). Fig. 3A shows the nuclear localization of activated GRs in dentate neurons. Fig. 3 A–F shows dentate gyrus sections collected at the end of FS (i.e., at 15 min) and Fig. 3G shows a section collected at 90 min. Using double immunofluorescence, we found colocalizations of pERK1/2 and pMSK1 (Fig. 3B), pERK1/2 and pElk-1 (Fig. 3C), pElk-1 and pMSK1 (Fig. 3D), pERK1/2 and H3S10p-K14ac (Fig. 3E), pERK1/2 and c-Fos (Fig. 3F), and c-Fos and Egr-1 (Fig. 3G). Previously, we demonstrated that H3S10p-K14ac and c-Fos colocalize in nuclei of dentate granule neurons (20). To ascertain whether the H3S10p-K14ac mark is associated with the c-fos gene promoter, we performed chromatin immunoprecipitation (ChIP) on hippocampus and, for control reasons, neocortex chromatin of rats killed under baseline conditions or 1 h after FS. Fig. 4 shows that FS results in the generation of H3S10p-K14ac within the c-fos promoter in hippocampus chromatin but not in neocortex chromatin. In addition, FS evoked a significant decline in histone H4 hyperacetylation (H4ac) but not in H3 hyperacetylation (H3ac) in the c-fos promoter in hippocampus and neocortex (Fig. 4).

Fig. 3.

Fig. 3.

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Immunofluorescence analysis of GR, ERK MAPK signaling partners and induced immediate-early gene products after stress. Rats were forced to swim and were perfused after 15 min (A–F) or 90 min (G), after which immunofluorescence analyses were conducted with combinations of the indicated primary antibodies. The figure shows representative images. Immunofluorescence of all tested molecules was nuclear except for that of pERK1/2, which was nuclear and cytoplasmic (B, C, E, and F). Colocalization of stained molecules is indicated by a yellow color in the merged images. Note that possibly due to differences in time courses of expression, some neurons may not show colocalization (for instance, E and G).

Fig. 4.

Fig. 4.

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Association of H3S10p-K14ac, H3ac, and H4ac with the c-fos promoter in hippocampus and neocortex chromatin under baseline conditions and at 1 h after forced swimming. Chromatin was extracted from hippocampus and neocortex tissues and digested using MNase. (A) Representative 1% agarose gel showing that MNase treatment of chromatin produced mainly mononucleosomes (∼150 bp DNA) and equivalently across tissues and treatment groups (Bs Neo. baseline neocortex; Fs Neo, forced swim neocortex; Bs Hip, baseline hippocampus; Fs Hip, forced swim hippocampus). ChIP was conducted on MNase-digested chromatin for the H3S10p-K14ac, H3ac, and H4ac marks. DNA was isolated and quantified as described in SI Materials and Methods. B and C show hippocampus and neocortex data, respectively, depicted as percentage of change to baseline and expressed as the mean of three independent experiments ± SEM. The baseline levels of c-fos promoter DNA after ChIP/real-time PCR on hippocampus chromatin were for the H3S10p-K14ac, H3ac, and H4ac marks 1.7 ± 0.2 ng, 17.3 ± 2.0 ng, and 20.3 ± 2.1 ng (mean ± SEM, n = 3), respectively. Neocortex chromatin: 1.4 ± 0.1 ng, 14.2 ± 0.4 ng, and 11.4 ± 1.4 ng (mean ± SEM, n = 3), respectively. *P < 0.05 compared with baseline, Student's t test.

GR Antagonist Attenuates MSK1 and Elk-1 Phosphorylation but Not ERK1/2 Phosphorylation After Stress.

To determine the role of GRs in the activation of ERK1/2, MSK1, and Elk-1 in dentate neurons after stress, rats received a single RU486 injection 30 min before a 15-min FS session and were killed immediately after. We also included pretreatments with the NMDA-R antagonist MK801 and the MR antagonist spironolactone. Previously, we reported that MK801 abolishes the FS-induced H3S10p-K14ac and c-Fos induction in dentate neurons and blocks the behavioral immobility response, whereas spironolactone is ineffective (11). Fig. 5 shows that MK801 strongly antagonizes the increases in pERK1/2, pMSK1, and pElk-1 in dentate neurons after stress (Fig. 5 A–C). Spironolactone had no effect (Fig. 5 A–C), which corresponds with its inability to affect H3S10p-K14ac, c-Fos, and behavior (11). RU486 however significantly decreased the stress-induced rises in pMSK1 and pElk-1 but did not affect the increase in pERK1/2 (Fig. 5 A–C). Thus, MSK1 and Elk-1 phosphorylation by pERK1/2 in dentate neurons requires activated GRs. RU486 had no effect on FS-induced pMSK1 in other hippocampal cells such as CA1 neurons (Fig. S3).

Fig. 5.

Fig. 5.

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Requirement of activated GRs for stress-induced MSK1 and Elk-1 phosphorylation (B and C) but not for stress-induced ERK1/2 phosphorylation (A). Rats were treated with vehicle (vh), the GR antagonist RU486 (RU), the NMDA-R antagonist MK801 (MK), or the MR antagonist spironolactone (Spi) and 30 min later forced to swim (FS) for 15 min, after which they were immediately perfused. Separate groups of rats, i.e., the baseline control groups (Bs), were treated with vehicle or drug and perfused 45 min later. pERK1/2, pMSK1, and pElk-1 in the dentate gyrus were visualized by immunohistochemistry. As there were no differences between drug-treated and vehicle-treated Bs animals, these data were pooled and presented as one “Bs” group. The graphs present group means ± SEM (n = 4–6 rats per group, except Bs: n = 11–12) of the average number of immunopositive neurons in a 50-μm section of the dentate gyrus. *P < 0.05, significantly different from the baseline group (Bs; Bonferroni-corrected post hoc tests with contrasts). #P < 0.05, significantly different from the vehicle-treated forced swim group (Vh/FS; Bonferroni-corrected post hoc tests with contrasts).

GR Forms a Complex with Activated ERK1/2 and MSK1.

Next, we investigated whether GR, pERK1/2, and pMSK1 physically interact after FS. As shown in Fig. 6A, FS increased the GR, pERK1/2, and pMSK1 concentration in the nuclear fraction (NF) prepared from hippocampus immediately after 15 min of FS. Immunoprecipitations (IPs) were performed on hippocampal NFs of rats killed at baseline or immediately after FS (Fig. 6 B–D). We found that IP of GRs resulted in the coimmunoprecipitation (co-IP) of both pERK1/2 and pMSK1 (Fig. 6B). Significantly stronger pERK1/2 and pMSK1 signals were found in NFs of stressed rats. IP of pERK1/2 or pMSK1 led to the co-IP of GR, mainly in NFs of stressed rats (Fig. 6 C and D). pERK1/2 IP also led to the pull-down of pMSK1 in NFs of stressed rats (Fig. 6C). Conversely, pMSK1 IP, however, resulted in hardly any co-IP of pERK1/2 (Fig. 6D). These observations suggest that GRs indeed interact with pERK1/2 and pMSK1 in hippocampal nuclei.

Fig. 6.

Fig. 6.

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Activated GRs directly interact with nuclear pERK1/2 and pMSK1. Rats were killed after 15-min FS or under baseline conditions after which the hippocampus was dissected and snap frozen in liquid N2. (A) Cytoplasmic (CF) and a nuclear fraction (NF) of hippocampus tissue processed for Western blotting for GR, pMSK1, pERK1/2, and GAPDH (input). Molecular weights (kilodaltons, kDa) are indicated at Left side of image. (B–D) Immunoprecipitation (IP) of GR, pERK1/2, and pMSK1 and analysis of co-IP'ed proteins in NFs of baseline (BsIP) and forced swim animals (FSIP). Input in B–D indicates the GAPDH content in the hippocampus nuclear fraction used for the corresponding IPs. A–D show representative Western blot images and quantification of the Western blot data. The quantitative data are expressed as percentage (%) of increase over the baseline value. *P < 0.05, one-sample Student's t test (n = 4–7).

Discussion

A single traumatic experience has long-term consequences for future behavioral responses to such events. Here we showed that the consolidation of stress-related, long-lasting behavioral responses depends critically on glucocorticoid hormones being released during the initial experience, which act via GRs in dentate gyrus neurons. In these neurons, activated GRs interact with NMDA-R–activated pERK1/2, resulting in MSK1 and Elk-1 activation, histone H3 phosphoacetylation, and c-Fos and Egr-1 induction. The interaction of GR with pERK1/2 occurred within 15 min and involved a physical contact between the two proteins. Thus, glucocorticoids exert long-lasting effects on stress-related behavioral responses via a unique, nongenomic action on ERK MAPK signaling leading to epigenetic modifications and gene expression in dentate gyrus granule neurons.

We used the FS test to model the effects of an acute traumatic experience on molecular changes in the brain and future behavioral responses related to the experience. Until now, however, it was unclear how activated GRs act in dentate gyrus neurons to enhance the consolidation of these behavioral responses (21, 26, 27). Recent work indicated that forcing rats or mice to swim results in H3S10p-K14ac and c-Fos induction in dentate neurons (19). Although these studies provided molecular endpoints (i.e., H3S10p-K14ac and c-Fos) for the action of glucocorticoids on a stress-induced behavioral response (11, 19), they did not give insight into the actual mechanism underlying the GR-mediated action. We postulated that after a FS challenge, activated GRs evoke epigenetic and gene expression changes and consolidation of behavioral immobility through a rapid interaction with the NMDA/ERK1/2/MSK1-signaling pathway (22). Activation of this signaling pathway would lead to serine-10 phosphorylation of histone H3 (by pMSK1) and acetylation of lysine-14, through recruitment of HATs, like CBP or p300. Here we found that c-Fos induction in sparse dentate granule neurons is indeed the result of ERK1/2 activation, MSK1 activation, and histone H3 phosphoacetylation of the c-fos promoter (Fig. S4). These neurons selectively express pElk-1, which most likely via p300 elicits the acetylation step in histone H3’s phosphoacetylation (15) (Fig. S4).

Stress resulted in Egr-1 induction in dentate granule neurons via signaling through GR and the ERK1/2–MSK1–Elk-1 pathways. The stress-induced elevations in dentate Egr-1 expression at 15 and 90 min correspond with the two previously reported waves of MAPK-independent and MAPK-dependent Egr-1 induction (24). Given the presence of Elk-1– and AP1-binding sites in the egr-1 gene promoter, the induction of Egr-1 at 90 min may be the consequence of Elk-1 activation and c-Fos induction (28). Previously, tetanic stimulation of the perforant path resulted in ERK MAPK-dependent Elk-1 phosphorylation, Egr-1 induction, and long-term potentiation in dentate neurons (29). We show that Egr-1 induction in dentate neurons after stress is the result of a concerted action of activated GRs, pElk-1, and possibly c-Fos. A direct role of pMSK1 and histone H3 phosphorylation in Egr-1 induction is presently unclear. Like c-Fos the expression of Egr-1 depends on NMDA-R activation and is required in synaptic plasticity and memory formation (30–32). The sequential activation of NMDA-Rs, ERK MAPK/MSK1/Elk-1 signaling, H3S10p-K14ac formation, and c-Fos induction in Egr-1 expression in dentate neurons suggests the requirement of sequential signaling and genomic mechanisms in memory consolidation (29, 33). The induction of Egr-1 in dentate neurons also requires glucocorticoid hormone action via GRs. The activated GRs do not act independently from the ERK MAPK pathway but actually interact with ERK1/2–MSK1–Elk-1 signaling.

On the basis of the colocalization of pERK1/2 with pMSK1, pElk-1, H3S10p-K14ac, and c-fos in dentate neurons, we addressed the question at which level GRs are interacting with the NMDA–ERK1/2–MSK1–Elk-1 pathway. Pretreatment with a GR antagonist inhibited the FS-induced increase in pMSK1 and pElk-1 but the response in pERK1/2 was intact. The NMDA antagonist attenuated responses in all three signaling molecules. Both antagonists block FS-induced H3S10p-K14ac, c-Fos, and Egr-1 induction in granule neurons (present study and refs. 11, 19), which is consistent with their effects on pMSK1 and pElk-1. The effect of GRs is fast because the GR antagonist inhibited the stress-induced responses in pMSK1 and pElk-1 within 15 min. Fast effects of glucocorticoids have been described to occur via cell membrane-associated GRs (34, 35). However, as the GR antagonist did not affect the stress-induced increases in pERK1/2 but decreased the stress-induced, pERK1/2-mediated rises in pMSK1 and pElk-1, we concluded that after stress, the generated pERK1/2 requires activated GRs for full kinase activity to phosphorylate MSK1 and Elk-1 (Fig. S4). Previously, glucocorticoid treatment of AtT-20 cells in vitro resulted in increased Ras, Raf-1, ERK1/2, and pERK1/2 levels after 1–3 h (24). This is a markedly slower effect than the responses in pERK1/2, pMSK1, and pElk-1 we observed after stress, which peaked at 15 min. Thus, the in vitro effects on pituitary cells are very different from our effects in neurons in vivo. Furthermore, treatment of rats with glucocorticoid hormones does not affect H3S10p-K14ac and c-Fos in dentate neurons (20). This can be explained by our observation that concurrently with GR activation, stress produces ERK1/2 activation to obtain full MSK1 and Elk-1 phosphorylation.

Co-IP studies showed that activated GRs form a complex with pERK1/2 and pMSK1. We could not demonstrate interactions with pElk-1 as this protein was masked by immunoglobulins on the Western blot. On the basis of our co-IP studies, activated GRs possibly act as a scaffold, enabling pERK1/2 to phosphorylate MSK1. Although the used hippocampal nuclear extracts may contain GRs, pERK1/2 and pMSK1 derived from other subregions such as CA1 (36), we found that stress-induced pMSK1 in the CA1 is not affected by GR antagonist treatment, indicating that the role of GRs in pERK1/2-mediated MSK1 phosphorylation is specific for dentate neurons. Thus, activated GRs enhance pERK1/2 kinase activity via a protein–protein interaction resulting in the phosphorylation of MSK1 and Elk-1 (Fig. S4). For full kinase activity pERK1/2 seems to require binding of other factors whose identity may depend on the type of cell and the physiological context. An in vitro study showed that pERK1/2 needs to bind the progesterone receptor (PR) to phosphorylate MSK1 enabling it to subsequently phosphorylate H3S10 (37). We found that a GR–pERK1/2 interaction is vital for the consolidation of long-lasting behavioral responses in the FS model. In other stress-related behavioral paradigms such as contextual fear conditioning and Morris water maze learning a role of hippocampal GRs and ERK MAPK signaling to MSK1 and histones have been identified (16–18). Therefore, also in these paradigms a direct action of GRs on pERK1/2 may be required for the activation of downstream signaling mechanisms, histone modifications, gene expression (e.g., c-Fos and Egr-1), and the consolidation of contextual fear and spatial memories of the endured events. This notion, however, needs to be experimentally verified.

In summary, glucocorticoid hormones released during a traumatic event activate GRs that in dentate neurons form complexes with concurrently activated pERK1/2, resulting in activation of histone-modifying enzymes, epigenetic changes, and induction of gene expression. These processes are associated with the consolidation of stress-related behavioral responses including memory formation of the adverse event. This unique, nongenomic mechanism of glucocorticoid action may provide an explanation why stressful events have a long-lasting impact on behavior and stress-related memories. These observations may be of significance for elucidating anxiety-related psychiatric disorders in which traumatic memories and associations play a principal role, such as posttraumatic stress disorder (38).

Materials and Methods

Animals and Drug Treatment.

Male Wistar rats (150-175 g) were purchased from Harlan and group housed. Rats were forced to swim for 15 min in 25 °C water or left undisturbed (11, 19). Some animals received pretreatment with a drug or the vehicle 30 min before FS. Rats were killed at the indicated times (see legends) after FS or were kept until 24 h or 4 wk later to undergo another FS test (retest) for 5 min. Behavior was scored every 10 s during the first 5 min of the test and retest. The used drugs were RU486 (100 mg/kg body weight), spironolactone (50 mg/kg), or MK801 (100 μg/kg, free base) to block GRs, MRs, and NMDA receptors, respectively. For more information, see SI Materials and Methods.

Tissue Preparation.

For immunohistochemistry rats were perfused with saline and 4% paraformaldehyde and inhibitors. Brains were cut into 50-μm coronal sections and kept at 4 °C. For other studies, after decapitation hippocampus and neocortex were rapidly dissected, snap frozen in liquid N2, and stored at −80 °C. For more information, see SI Materials and Methods.

Immunohistochemistry.

For immunohistochemistry, see SI Materials and Methods.

ChIP and Real-Time PCR.

ChIP was performed using a published method (39). For a complete description, see SI Materials and Methods.

Co-IP and Western Blot Analysis.

Nuclear and cytoplasmic fractions were prepared from hippocampus tissue of rats. Samples were separated by SDS/PAGE and blotted to a membrane. For more information, see SI Materials and Methods.

Statistical Analysis.

For statistical analysis, see SI Materials and Methods.

Supplementary Material

Supporting Information
supp_108_33_13806__index.html (1.1KB, html)

Acknowledgments

We thank Dr. K. R. Mifsud, Ms. E. Saunderson, Ms. R. Demski-Allen, and Dr. X. Qian for their help with the experiments. This work was supported by the Biotechnology and Biological Sciences Research Council (Grants BB/F000510/1 and BB/G02507X/1).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1104383108/-/DCSupplemental.

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