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

María Gutièrrez-Mecinas, Alexandra F. Trollope, Andrew Collins, Hazel Morfett, Shirley A. Hesketh, Flavie Kersanté, and Johannes M. H. M. Reul1

Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom

Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved July 6, 2011 (received for review April 8, 2011)

Stressful events are known to have a long-term impact on future promoters (e.g., c-fos) (12, 13). pElk-1 binds to the serum behavioral stress responses. Previous studies suggested that both response element (SRE) in distinct gene promoters [including glucocorticoid hormones and glutamate acting via glucocorticoid the c-fos and egr-1 (early growth response protein 1) promoters], receptors (GRs) and N-methyl D-aspartate (NMDA) receptors, respec- after which it recruits the histone acetyl transferase (HAT) p300 tively, are of critical importance for the consolidation of these long- (15). Thus, MSK1 and Elk-1 activation results in histone H3 lasting behavioral responses at the dentate gyrus, the gateway of phosphorylation and acetylation in gene promoters, thereby ac- the hippocampal formation. We found that an acute psychologically tivating gene . stressful event resulted in ERK1/2 phosphorylation (pERK1/2), Blockade of NMDA-Rs, inhibition of ERK1/2 activation, and which within 15 min led to the activation of the nuclear kinases gene deletion of MSK1 in vivo prevent histone H3 phosphory- MSK1 and Elk-1 in granule neurons of the dentate gyrus. Next, lation and acetylation and c-Fos induction in the and impair behavioral responses in the Morris water maze, MSK1 and Elk-1 activation evoked serine-10 phosphorylation and contextual fear conditioning, and forced swim test (11, 16). lysine-14 acetylation in histone H3, resulting in the induction of the Furthermore, glucocorticoid hormones secreted as a result of the neuroplasticity-associated immediate-early c-Fos and Egr-1 in behavioral challenge are of great importance in Morris water

these neurons. The pERK1/2-mediated activation of MSK1 and Elk-1 NEUROSCIENCE – maze learning and contextual fear conditioning (17, 18). More- required a rapid protein protein interaction between pERK1/2 and over, epigenetic, , and behavioral responses to activated GRs. This is a unique nongenomic mechanism of glucocor- forced swimming (FS) and novelty critically depend on GR- ticoid hormone action in dentate gyrus granule neurons on long- mediated glucocorticoid action in dentate gyrus granule neurons lasting behavioral responses to stress involving direct cross-talk of (19–21). Thus, stress-induced behavioral responses involve acti- GRs with ERK1/2–MSK1–Elk-1 signaling to the nucleus. vation of ERK MAPK and GR-mediated signaling in dentate neurons that converge and result in specific histone H3 mod- | chromatin | epigenetics | hippocampus | memory ifications [i.e., serine-10 (S10) phosphorylation and lysine-14 (K14) acetylation (H3S10p-K14ac)] and activation of gene tran- drenal glucocorticoid hormones play an important role in scription (22). How these signaling pathways converge is pres- Athe behavioral consequences of stress (1). ently unknown. We postulated that glucocorticoids enhance secreted during a stressful event facilitate learning of adaptive molecular and behavioral responses through a direct interaction behavioral responses and the consolidation of memories of the of GRs with ERK1/2 and its downstream partners, MSK1 and event (1, 2). Aberrant glucocorticoid secretion, as a result of Elk-1, in dentate gyrus neurons. chronic stress, is implicated in stress-related disorders such as We show that after an acute stressful challenge glucocorticoid major depression and anxiety (3–5). hormones via GRs rapidly enhance the pERK1/2-mediated It is still unclear how glucocorticoid hormones affect behavior generation of pMSK1 and pElk-1, leading to the phosphoryla- at the molecular level. Glucocorticoid levels attained after stress tion and acetylation of histone H3 and induction of c-Fos and fi influence cellular function by activating glucocorticoid receptors Egr-1 speci cally in dentate neurons. Furthermore, GRs en- (GRs) (6). These receptors bind to their target sites in gene hance ERK MAPK signaling to the chromatin and behavior promoters, thereby changing gene expression (7). Activated GRs through a physical interaction with pERK1/2 and pMSK1. can also interact through protein–protein interactions with a broad range of intracellular signaling molecules including tran- Results scription factors and enzymes (7). Whether GRs directly interact Long-Term Glucocorticoid Effects on a Stress-Related Behavioral with intracellular signaling pathways to influence stress-related Response. Fig. 1A shows that when rats are subjected to an behavior is unknown. acute FS challenge (i.e., 15 min in 25 °C water) they display, A signaling pathway involved in behavioral adaptation and upon reexposure to the challenge 24 h later, substantially more memory formation is the extracellular signal-regulated kinase immobility behavior than during the initial test. Fig. 1B shows mitogen-activated protein kinase (ERK MAPK) signaling path- that this behavioral response is maintained not just for 24 h but way (8). This pathway is activated through N-methyl D-aspartate actually for at least 4 wk. The consolidation of this long-lasting receptors (NMDA-Rs) and other membrane receptors and is behavioral response requires an action of glucocorticoid hor- involved in changes in neuronal structure and function (8). mones via GRs, but not mineralocorticoid receptors (MRs), in Hippocampal NMDA-R–mediated ERK MAPK signaling is in- volved in behavioral responses observed in Morris water maze learning, contextual fear conditioning, and the forced swim test Author contributions: M.G.-M. and J.M.H.M.R. designed research; M.G.-M., A.F.T., A.C., (9–11). In vitro experiments suggest that ERK MAPK signaling H.M., S.A.H., F.K., and J.M.H.M.R. performed research; M.G.-M., A.F.T., and J.M.H.M.R. activates nuclear histone modifying enzymes such as MSK1 analyzed data; and M.G.-M., A.F.T., and J.M.H.M.R. wrote the paper. (mitogen- and stress-activated kinase 1) (12, 13) and Elk-1 (ETS The authors declare no conflict of interest. domain protein-1) (14). These enzymes evoke changes in the This article is a PNAS Direct Submission. chromatin structure underlying gene transcription. Both kinases 1To whom correspondence should be addressed. E-mail: [email protected]. – are substrates of ERK1/2 (12 14). Phosphorylated MSK1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (pMSK1) phosphorylates serine-10 in histone H3 tails in various 1073/pnas.1104383108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1104383108 PNAS Early Edition | 1of6 Downloaded by guest on October 4, 2021 Fig. 1. Effect of the GR antagonist RU486 on FS- induced behavioral responses (A and B) and mo- lecular 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 behav- ior 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 re- peated 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) anti- bodies. 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.

the dentate gyrus during the initial test (21). Accordingly, GR (6, 25). Fig. 3A shows the nuclear localization of activated GRs in antagonist (e.g., RU486) injection before the initial test results in dentate neurons. Fig. 3 A–F shows dentate gyrus sections collected an impaired behavioral immobility response in a retest 24 h later at the end of FS (i.e., at 15 min) and Fig. 3G shows a section col- but also if tested 4 wk later (Fig. 1 A and B). lected at 90 min. Using double immunofluorescence, we found Fig. 1 C–E shows that during the consolidation phase that colocalizations of pERK1/2 and pMSK1 (Fig. 3B), pERK1/2 and follows the initial FS challenge H3S10p-K14ac, c-Fos, and Egr-1 pElk-1 (Fig. 3C), pElk-1 and pMSK1 (Fig. 3D), pERK1/2 and are generated in dentate neurons. These responses occurred H3S10p-K14ac (Fig. 3E), pERK1/2 and c-Fos (Fig. 3F), and c-Fos selectively in mature granule neurons of the dorsal blade (Fig. and Egr-1 (Fig. 3G). Previously, we demonstrated that H3S10p- S1). Pretreatment with RU486 blocked the FS-induced respon- K14ac and c-Fos colocalize in nuclei of dentate granule neurons ses in H3S10p-K14ac c-Fos, and Egr-1 (Fig. 1 C–E). (20). To ascertain whether the H3S10p-K14ac mark is associated with the c-fos gene promoter, we performed chromatin immuno- Stress Evokes Phosphorylation of ERK1/2, MSK1, and Elk-1 in Dentate precipitation (ChIP) on hippocampus and, for control reasons, Gyrus Granule Neurons. We investigated the activation of ERK1/2 neocortex chromatin of rats killed under baseline conditions or 1 h and its downstream substrates MSK1 and Elk-1 by immunohis- after FS. Fig. 4 shows that FS results in the generation of H3S10p- tochemical analyses of pERK1/2, pMSK1, pElk-1, H3S10p- K14ac within the c-fos promoter in hippocampus chromatin but K14ac, c-Fos, and Egr-1 in dentate neurons after FS (Fig. 2). The not in neocortex chromatin. In addition, FS evoked a significant immunoreactive proteins presented a sparse staining pattern in decline in histone H4 hyperacetylation (H4ac) but not in H3 dentate neurons. pERK1/2 staining was found in both the nu- hyperacetylation (H3ac) in the c-fos promoter in hippocampus clear and cytoplasmic compartments (Fig. 2A), whereas staining and neocortex (Fig. 4). of pMSK1, pElk-1, H3S10p-K14ac, c-Fos, and Egr-1 was only nuclear (Fig. 2 B–F). Furthermore, the stress-induced signaling GR Antagonist Attenuates MSK1 and Elk-1 Phosphorylation but Not was specific through pERK1/2 and pMSK1 as neither p- ERK1/2 Phosphorylation After Stress. To determine the role of GRs p38MAPK (another MSK1 kinase) nor pRSK1/2 (an MSK-like in the activation of ERK1/2, MSK1, and Elk-1 in dentate neu- kinase) (23) was found after stress. The phosphorylation of rons after stress, rats received a single RU486 injection 30 min ERK1/2, MSK1, and Elk-1 was a rapid, transient phenomenon before a 15-min FS session and were killed immediately after. peaking at 15 min after start of the FS challenge (Fig. 2 A–C). We also included pretreatments with the NMDA-R antagonist Fig. 1 D and E and previous work (11) show that H3S10p-K14ac MK801 and the MR antagonist spironolactone. Previously, we and c-Fos peak at 1–2 h and return to baseline at 4 h (11). The reported that MK801 abolishes the FS-induced H3S10p-K14ac increased expression of pERK1/2, pMSK1, and pElk-1 after FS and c-Fos induction in dentate neurons and blocks the behav- occurred only in mature dentate neurons of the dorsal blade ioral immobility response, whereas spironolactone is ineffective (Fig. S2). Egr-1 levels were elevated at 15 min and 90 min after (11). Fig. 5 shows that MK801 strongly antagonizes the increases FS (Fig. 2F). The fast increase after stress (i.e., at 15 min) has in pERK1/2, pMSK1, and pElk-1 in dentate neurons after stress been reported to be MAPK independent, whereas the second (Fig. 5 A–C). Spironolactone had no effect (Fig. 5 A–C), which increase at 90 min is MAPK dependent (24). The rise in Egr-1 at corresponds with its inability to affect H3S10p-K14ac, c-Fos, and 15 min was found in young and mature dentate neurons of both behavior (11). RU486 however significantly decreased the stress- dorsal and ventral blades, whereas the increase at 90 min was induced rises in pMSK1 and pElk-1 but did not affect the in- only observed in mature dorsal blade neurons (Fig. S1). crease in pERK1/2 (Fig. 5 A–C). Thus, MSK1 and Elk-1 phos- phorylation by pERK1/2 in dentate neurons requires activated Colocalization of pERK1/2, pMSK1, pElk-1, H3S10p-K14ac, c-Fos, and GRs. RU486 had no effect on FS-induced pMSK1 in other Egr-1 in Dentate Gyrus Granule Neurons. To study glucocorticoid hippocampal cells such as CA1 neurons (Fig. S3). interaction with NMDA-ERK MAPK signaling, colocalization of pERK1/2, pMSK1, and pElk-1 in dentate neurons needed to be GR Forms a Complex with Activated ERK1/2 and MSK1. Next, we in- demonstrated first. Dentate neurons express GRs and NMDA-Rs vestigated whether GR, pERK1/2, and pMSK1 physically interact

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Fig. 2. 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).

after FS. As shown in Fig. 6A, FS increased the GR, pERK1/2, and dentate gyrus neurons. In these neurons, activated GRs in- pMSK1 concentration in the nuclear fraction (NF) prepared from teract with NMDA-R–activated pERK1/2, resulting in MSK1 hippocampus immediately after 15 min of FS. Immunoprecipita- and Elk-1 activation, histone H3 phosphoacetylation, and c-Fos tions (IPs) were performed on hippocampal NFs of rats killed at and Egr-1 induction. The interaction of GR with pERK1/2 baseline or immediately after FS (Fig. 6 B–D). We found that IP occurred within 15 min and involved a physical contact be- of GRs resulted in the coimmunoprecipitation (co-IP) of both tween the two proteins. Thus, glucocorticoids exert long-lasting pERK1/2 and pMSK1 (Fig. 6B). Significantly stronger pERK1/2 effects on stress-related behavioral responses via a unique, and pMSK1 signals were found in NFs of stressed rats. IP of nongenomic action on ERK MAPK signaling leading to epi- pERK1/2 or pMSK1 led to the co-IP of GR, mainly in NFs of genetic modificationsandgeneexpressionindentategyrus stressed rats (Fig. 6 C and D). pERK1/2 IP also led to the pull- granule neurons. down of pMSK1 in NFs of stressed rats (Fig. 6C). Conversely, We used the FS test to model the effects of an acute traumatic pMSK1 IP, however, resulted in hardly any co-IP of pERK1/2 (Fig. experience on molecular changes in the brain and future be- 6D). These observations suggest that GRs indeed interact with havioral responses related to the experience. Until now, how- pERK1/2 and pMSK1 in hippocampal nuclei. ever, it was unclear how activated GRs act in dentate gyrus neurons to enhance the consolidation of these behavioral Discussion responses (21, 26, 27). Recent work indicated that forcing rats or A single traumatic experience has long-term consequences for mice to swim results in H3S10p-K14ac and c-Fos induction in future behavioral responses to such events. Here we showed dentate neurons (19). Although these studies provided molecular that the consolidation of stress-related, long-lasting behavioral endpoints (i.e., H3S10p-K14ac and c-Fos) for the action of glu- responses depends critically on glucocorticoid hormones being cocorticoids on a stress-induced behavioral response (11, 19), released during the initial experience, which act via GRs in they did not give insight into the actual mechanism underlying

Gutièrrez-Mecinas et al. PNAS Early Edition | 3of6 Downloaded by guest on October 4, 2021 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 path- ways. 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 in- duction (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 syn- aptic 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 expres- sion 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 actu- ally 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 ad- dressed 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 -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, glucocor- ticoid 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 Fig. 3. Immunofluorescence analysis of GR, ERK MAPK signaling partners is a markedly slower effect than the responses in pERK1/2, and induced immediate-early gene products after stress. Rats were forced to pMSK1, and pElk-1 we observed after stress, which peaked at 15 swim and were perfused after 15 min (A–F) or 90 min (G), after which im- min. Thus, the in vitro effects on pituitary cells are very different munofluorescence analyses were conducted with combinations of the in- from our effects in neurons in vivo. Furthermore, treatment of dicated primary antibodies. The figure shows representative images. rats with glucocorticoid hormones does not affect H3S10p-K14ac Immunofluorescence of all tested molecules was nuclear except for that of and c-Fos in dentate neurons (20). This can be explained by our pERK1/2, which was nuclear and cytoplasmic (B, C, E, and F). Colocalization observation that concurrently with GR activation, stress pro- of stained molecules is indicated by a yellow color in the merged images. duces ERK1/2 activation to obtain full MSK1 and Elk-1 phos- Note that possibly due to differences in time courses of expression, some phorylation. neurons may not show colocalization (for instance, E and G). 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 GR-mediated action. We postulated that after a FS chal- the Western blot. On the basis of our co-IP studies, activated lenge, activated GRs evoke epigenetic and gene expression GRs possibly act as a scaffold, enabling pERK1/2 to phosphor- changes and consolidation of behavioral immobility through ylate MSK1. Although the used hippocampal nuclear extracts a rapid interaction with the NMDA/ERK1/2/MSK1-signaling may contain GRs, pERK1/2 and pMSK1 derived from other pathway (22). Activation of this signaling pathway would lead to subregions such as CA1 (36), we found that stress-induced serine-10 phosphorylation of histone H3 (by pMSK1) and acet- pMSK1 in the CA1 is not affected by GR antagonist treatment, ylation of lysine-14, through recruitment of HATs, like CBP or indicating that the role of GRs in pERK1/2-mediated MSK1 p300. Here we found that c-Fos induction in sparse dentate phosphorylation is specific for dentate neurons. Thus, activated granule neurons is indeed the result of ERK1/2 activation, GRs enhance pERK1/2 kinase activity via a protein–protein in- MSK1 activation, and histone H3 phosphoacetylation of the c-fos teraction resulting in the phosphorylation of MSK1 and Elk-1 promoter (Fig. S4). These neurons selectively express pElk-1, (Fig. S4). For full kinase activity pERK1/2 seems to require

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1104383108 Gutièrrez-Mecinas et al. Downloaded by guest on October 4, 2021 Fig. 4. Association of H3S10p-K14ac, H3ac, and H4ac with the c-fos promoter in hippocampus and neocortex chromatin under baseline con- ditions and at 1 h after forced swimming. Chro- matin 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.

binding of other factors whose identity may depend on the type contextual fear conditioning and Morris water maze learning of cell and the physiological context. An in vitro study showed a role of hippocampal GRs and ERK MAPK signaling to MSK1 that pERK1/2 needs to bind the (PR) to and histones have been identified (16–18). Therefore, also in phosphorylate MSK1 enabling it to subsequently phosphorylate these paradigms a direct action of GRs on pERK1/2 may be H3S10 (37). We found that a GR–pERK1/2 interaction is vital required for the activation of downstream signaling mechanisms, for the consolidation of long-lasting behavioral responses in the histone modifications, gene expression (e.g., c-Fos and Egr-1), FS model. In other stress-related behavioral paradigms such as and the consolidation of contextual fear and spatial memories of the endured events. This notion, however, needs to be ex- perimentally verified.

In summary, glucocorticoid hormones released during a trau- NEUROSCIENCE matic event activate GRs that in dentate neurons form com- plexes with concurrently activated pERK1/2, resulting in ac- tivation of histone-modifying enzymes, epigenetic changes, and induction of gene expression. These processes are associated with the consolidation of stress-related behavioral responses in- cluding 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 dis- orders 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 Fig. 5. Requirement of activated GRs for stress-induced MSK1 and Elk-1 sections and kept at 4 °C. For other studies, after decapitation hippocampus phosphorylation (B and C) but not for stress-induced ERK1/2 phosphorylation and neocortex were rapidly dissected, snap frozen in liquid N2, and stored (A). Rats were treated with vehicle (vh), the GR antagonist RU486 (RU), the at −80 °C. For more information, see SI Materials and Methods. 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 im- Immunohistochemistry. For immunohistochemistry, see SI Materials and Methods. mediately 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 immunohisto- ChIP and Real-Time PCR. ChIP was performed using a published method chemistry. As there were no differences between drug-treated and vehicle- (39). For a complete description, see SI Materials and Methods. 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 = Co-IP and Western Blot Analysis. Nuclear and cytoplasmic fractions were 11–12) of the average number of immunopositive neurons in a 50-μm section prepared from hippocampus tissue of rats. Samples were separated by SDS/ of the dentate gyrus. *P < 0.05, significantly different from the baseline group PAGE and blotted to a membrane. For more information, see SI Materials (Bs; Bonferroni-corrected post hoc tests with contrasts). #P < 0.05, significantly and Methods. different from the vehicle-treated forced swim group (Vh/FS; Bonferroni- corrected post hoc tests with contrasts). Statistical Analysis. For statistical analysis, see SI Materials and Methods.

Gutièrrez-Mecinas et al. PNAS Early Edition | 5of6 Downloaded by guest on October 4, 2021 Fig. 6. 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) Cyto- plasmic (CF) and a nuclear fraction (NF) of hippocampus tissue processed for Western blotting for GR, pMSK1, pERK1/2, and GAPDH (input). Molecular weights (kilo- daltons, 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 nu- clear 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).

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

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