Background: Although glucocorticoid receptors (GRs) in the hippocampus play a vital role in the regulation of physiological and behavioural responses to stress, the regulation of receptor expression remains unclear. This work investigates the molecular mechanisms underpinning stress-induced changes in hippocampal GR mRNA levels in vivo. Methods: Male Wistar rats were killed either under baseline conditions or after forced swim stress (FSS; 15 min in 25°C water). Rat hippocampi were micro-dissected (for mRNA, microRNA, and DNA methylation analysis) or frozen whole (for chromatin immunoprecipitation). In an additional experiment, rats were pre-treated with RU486 (a GR antagonist) or vehicle. Results: FSS evoked a dentate gyrus-specific reduction in GR mRNA levels. This was related to an increased DNMT3a protein association with a discreet region of the Nr3c1 (GR gene) promoter, shown here to undergo increased DNA methylation after FSS. FSS also caused a time-dependent increase in the expression of miR-124a, a microRNA known to reduce GR mRNA expression, which was inversely correlated with a reduction in GR mRNA levels 30 min after FSS. FSS did not affect GR binding to a putative negative glucocorticoid response element within the Nr3c1 gene. Conclusions: Acute stress results in decreased GR mRNA expression specifically in the dentate gyrus. Our results indicate that a complex interplay of multiple molecular mechanisms - including increased DNA methylation of discrete CpG residues within the Nr3c1 gene, most likely facilitated by DNMT3a, and increased expression of miR-124a - could be responsible for these changes.

Reul JMHM: Making memories of stressful events: a journey along epigenetic, gene transcription, and signaling pathways. Front Psychiatry 2014;5:5.
Reul JMHM, Collins A, Saliba RS, Mifsud KR, Carter SD, Gutièrrez-Mecinas M, et al: Glucocorticoids, epigenetic control and stress resilience. Neurobiol Stress 2015;1:44-59.
Gutièrrez-Mecinas M, Trollope AF, Collins A, Morfett H, Hesketh SA, Kersanté F, et al: Long-lasting behavioral responses to stress involve a direct interaction of glucocorticoid receptors with ERK1/2-MSK1-Elk-1 signaling. Proc Natl Acad Sci USA 2011;108:13806-13811.
Reul JMHM, de Kloet ER: Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 1985;117:2505-2511.
Reul JMHM, van den Bosch FR, de Kloet ER: Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J Endocrinol 1987;115:459-467.
Reul JMHM, de Kloet ER: Anatomical resolution of two types of corticosterone receptor sites in rat brain with in vitro autoradiography and computerized image analysis. J Steroid Biochem 1986;24:269-272.
Datson NA, Meijer L, Steenbergen PJ, Morsink MC, van der Laan S, Meijer OC, et al: Expression profiling in laser-microdissected hippocampal subregions in rat brain reveals large subregion-specific differences in expression. Eur J Neurosci 2004;20:2541-2554.
Droste SK, Chandramohan Y, Hill LE, Linthorst AC, Reul JMHM: Voluntary exercise impacts on the rat hypothalamic-pituitary- adrenocortical axis mainly at the adrenal level. Neuroendocrinology 2007;86:26-37.
Sarabdjitsingh RA, Meijer OC, Schaaf MJ, de Kloet ER: Subregion-specific differences in translocation patterns of mineralocorticoid and glucocorticoid receptors in rat hippocampus. Brain Res 2009;1249:43-53.
Karandrea D, Kittas C, Kitraki E: Forced swimming differentially affects male and female brain corticosteroid receptors. Neuroendocrinology 2002;75:217-226.
Veenema AH, Meijer OC, de Kloet ER, Koolhaas JM, Bohus BG: Differences in basal and stress-induced HPA regulation of wild house mice selected for high and low aggression. Horm Behav 2003;43:197-204.
Aguilar-Valles A, Sánchez E, de Gortari P, Balderas I, Ramírez-Amaya V, Bermúdez-Rattoni F, et al: Analysis of the stress response in rats trained in the water-maze: differential expression of corticotropin-releasing hormone, CRH-R1, glucocorticoid receptors and brain-derived neurotrophic factor in limbic regions. Neuroendocrinology 2005;82:306-319.
Uchida S, Nishida A, Hara K, Kamemoto T, Suetsugi M, Fujimoto M, et al: Characterization of the vulnerability to repeated stress in Fischer 344 rats: possible involvement of microRNA-mediated down-regulation of the glucocorticoid receptor. Eur J Neurosci 2008;27:2250-2261.
Witzmann SR, Turner JD, Mériaux SB, Meijer OC, Muller CP: Epigenetic regulation of the glucocorticoid receptor promoter 17 in adult rats. Epigenetics 2012;7:1290-1301.
Paskitti ME, McCreary BJ, Herman JP: Stress regulation of adrenocorticosteroid receptor gene transcription and mRNA expression in rat hippocampus: time-course analysis. Brain Res Mol Brain Res 2000;80:142-152.
Nishimura K, Makino S, Tanaka Y, Kaneda T, Hashimoto K: Altered expression of p53 mRNA in the brain and pituitary during repeated immobilization stress: negative correlation with glucocorticoid receptor mRNA levels. J Neuroendocrinol 2004;16:84-91.
McCormick JA, Lyons V, Jacobson MD, Noble J, Diorio J, Nyirenda M, et al: 5′-heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events. Mol Endocrinol 2000;14:506-517.
Weaver IC, Cervoni N, Champagne FA, D'Alessio AC, Sharma S, Seckl JR, et al: Epigenetic programming by maternal behavior. Nat Neurosci 2004;7:847-854.
Jones MW, Errington ML, French PJ, Fine A, Bliss TV, Garel S, et al: A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat Neurosci 2001;4:289-296.
McGowan PO, Sasaki A, D'Alessio AC, Dymov S, Labonte B, Szyf M, et al: Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse. Nat Neurosci 2009;12:342-348.
van der Knaap LJ, Riese H, Hudziak JJ, Verbiest MM, Verhulst FC, Oldehinkel AJ, et al: Glucocorticoid receptor gene (NR3C1) methylation following stressful events between birth and adolescence. The TRAILS study. Transl Psychiatry 2014;4:e381.
Turecki G, Meaney MJ: Effects of the social environment and stress on glucocorticoid receptor gene methylation: a systematic review. Biol Psychiatry 2014;79:87-96.
Vreugdenhil E, Verissimo CS, Mariman R, Kamphorst JT, Barbosa JS, Zweers T, et al: MicroRNA 18 and 124a down-regulate the glucocorticoid receptor: implications for glucocorticoid responsiveness in the brain. Endocrinology 2009;150:2220-2228.
Durairaj RV, Koilmani ER: Environmental enrichment modulates glucocorticoid receptor expression and reduces anxiety in Indian field male mouse Mus booduga through up-regulation of microRNA-124a. Gen Comp Endocrinol 2014;199:26-32.
Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T: Identification of tissue-specific microRNAs from mouse. Curr Biol 2002;12:735-739.
Shimizu S, Tanaka T, Tohyama M, Miyata S: Yokukansan normalizes glucocorticoid receptor protein expression in oligodendrocytes of the corpus callosum by regulating microRNA-124a expression after stress exposure. Brain Res Bull 2015;114:49-55.
Ramamoorthy S, Cidlowski JA: Ligand-induced repression of the glucocorticoid receptor gene is mediated by an NCoR1 repression complex formed by long-range chromatin interactions with intragenic glucocorticoid response elements. Mol Cell Biol 2013;33:1711-1722.
Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.
Hoeck W, Rusconi S, Groner B: Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells. Investigations with a monospecific antiserum against a bacterially expressed receptor fragment. J Biol Chem 1989;264:14396-14402.
Cadepond F, Ulmann A, Baulieu EE: RU486 (mifepristone): mechanisms of action and clinical uses. Annu Rev Med 1997;48:129-156.
Saunderson EA, Spiers H, Mifsud KR, Gutierrez-Mecinas M, Trollope AF, Shaikh A, et al: Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus. Proc Natl Acad Sci USA 2016, Epub ahead of print.
Irvine RA, Lin IG, Hsieh CL: DNA methylation has a local effect on transcription and histone acetylation. Mol Cell Biol 2002;22:6689-6696.
Weaver IC: Epigenetic programming by maternal behavior and pharmacological intervention. Nature versus nurture: let's call the whole thing off. Epigenetics 2007;2:22-28.
Weaver IC, Diorio J, Seckl JR, Szyf M, Meaney MJ: Early environmental regulation of hippocampal glucocorticoid receptor gene expression: characterization of intracellular mediators and potential genomic target sites. Ann NY Acad Sci 2004;1024:182-212.
Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281-297.
De Kloet ER, Reul JMHM: Feedback action and tonic influence of corticosteroids on brain function: a concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology 1987;12:83-105.
Tran L, Chaloner A, Sawalha AH, Greenwood Van-Meerveld B: Importance of epigenetic mechanisms in visceral pain induced by chronic water avoidance stress. Psychoneuroendocrinology 2013;38:898-906.
Webster MJ, Knable MB, O'Grady J, Orthmann J, Weickert CS: Regional specificity of brain glucocorticoid receptor mRNA alterations in subjects with schizophrenia and mood disorders. Mol Psychiatry 2002;7:985-994.
McIntyre WR, Samuels HH: Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form. J Biol Chem 1985;260:418-427.
Nordeen SK, Bona BJ, Beck CA, Edwards DP, Borror KC, DeFranco DB: The two faces of a steroid antagonist: when an antagonist isn't. Steroids 1995;60:97-104.
Roberson ED, English JD, Adams JP, Selcher JC, Kondratick C, Sweatt JD: The mitogen-activated protein kinase cascade couples PKA and PKC to cAMP response element binding protein phosphorylation in area CA1 of hippocampus. J Neurosci 1999;19:4337-4348.
Perroud N, Rutembesa E, Paoloni-Giacobino A, Mutabaruka J, Mutesa L, Stenz L, et al: The Tutsi genocide and transgenerational transmission of maternal stress: epigenetics and biology of the HPA axis. World J Biol Psychiatry 2014;15:334-345.
Chandramohan Y, Droste SK, Reul JMHM: Novelty stress induces phospho-acetylation of histone H3 in rat dentate gyrus granule neurons through coincident signalling via the N-methyl-D-aspartate receptor and the glucocorticoid receptor: relevance for c-fos induction. J Neurochem 2007;101:815-828.
Chandramohan Y, Droste SK, Arthur JS, Reul JMHM: The forced swimming-induced behavioural immobility response involves histone H3 phospho-acetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/mitogen- and stress-activated kinase signalling pathway. Eur J Neurosci 2008;27:2701-2713.
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