The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice.

Disturbed activation or regulation of the stress response through the hypothalamic-pituitary-adrenal (HPA) axis is a fundamental component of multiple stress-related diseases, including psychiatric, metabolic, and immune disorders. The FK506 binding protein 51 (FKBP5) is a negative regulator of the glucocorticoid receptor (GR), the main driver of HPA axis regulation, and FKBP5 polymorphisms have been repeatedly linked to stress-related disorders in humans. However, the specific role of Fkbp5 in the paraventricular nucleus of the hypothalamus (PVN) in shaping HPA axis (re)activity remains to be elucidated. We here demonstrate that the deletion of Fkbp5 in Sim1+ neurons dampens the acute stress response and increases GR sensitivity. In contrast, Fkbp5 overexpression in the PVN results in a chronic HPA axis over-activation, and a PVN-specific rescue of Fkbp5 expression in full Fkbp5 KO mice normalizes the HPA axis phenotype. Single-cell RNA sequencing revealed the cell-type-specific expression pattern of Fkbp5 in the PVN and showed that Fkbp5 expression is specifically upregulated in Crh+ neurons after stress. Finally, Crh-specific Fkbp5 overexpression alters Crh neuron activity, but only partially recapitulates the PVN-specific Fkbp5 overexpression phenotype. Together, the data establish the central and cell-type-specific importance of Fkbp5 in the PVN in shaping HPA axis regulation and the acute stress response.

Acute stress enhances heterodimerization and binding of corticosteroid receptors at glucocorticoid target genes in the hippocampus.

A stressful event results in secretion of glucocorticoid hormones, which bind to mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the hippocampus to regulate cognitive and affective responses to the challenge. MRs are already highly occupied by low glucocorticoid levels under baseline conditions, whereas GRs only become substantially occupied by stress- or circadian-driven glucocorticoid levels. Currently, however, the binding of MRs and GRs to glucocorticoid-responsive elements (GREs) within hippocampal glucocorticoid target genes under such physiological conditions in vivo is unknown. We found that forced swim (FS) stress evoked increased hippocampal RNA expression levels of the glucocorticoid-responsive genes FK506-binding protein 5 (Fkbp5), Period 1 (Per1), and serum- and glucocorticoid-inducible kinase 1 (Sgk1). Chromatin immunoprecipitation (ChIP) analysis showed that this stressor caused substantial gene-dependent increases in GR binding and surprisingly, also MR binding to GREs within these genes. Different acute challenges, including novelty, restraint, and FS stress, produced distinct glucocorticoid responses but resulted in largely similar MR and GR binding to GREs. Sequential and tandem ChIP analyses showed that, after FS stress, MRs and GRs bind concomitantly to the same GRE sites within Fkbp5 and Per1 but not Sgk1 Thus, after stress, MRs and GRs seem to bind to GREs as homo- and/or heterodimers in a gene-dependent manner. MR binding to GREs at baseline seems to be restricted, whereas after stress, GR binding may facilitate cobinding of MR. This study reveals that the interaction of MRs and GRs with GREs within the genome constitutes an additional level of complexity in hippocampal glucocorticoid action beyond expectancies based on ligand-receptor interactions.

Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus.

Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5'-cytosine-phosphate-guanine-3') sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental stimuli in terms of gene expression and behavior.

Mineralocorticoid and glucocorticoid receptor-mediated control of genomic responses to stress in the brain.

Successful coping with stressful events involves adaptive and cognitive processes in the brain that make the individual more resilient to similar stressors in the future. Stressful events result in the secretion of glucocorticoids (GCs) from the adrenal glands into the blood stream. Early work proved instrumental for developing the concept that these hormones act in the brain to coordinate physiological and behavioral responses to stress through binding to two different GC-binding receptors. Once activated these receptors translocate to the nucleus where they act on target genes to facilitate (or sometimes inhibit) transcription. There are two types of receptors in the brain, the mineralocorticoid receptor (MR), and glucocorticoid receptor (GR). This review summarizes recent work which provides new insights regarding the genomic action of these receptors, both under baseline conditions and following exposure to acute stress. This work is discussed alongside the extensive studies undertaken in this field previously and new, and exciting "big data" studies which have generated a wealth of relevant data. The consequence of these new insights will challenge existing assumptions about the role of MRs and GRs and pave the way for the implementation of novel and improved methodologies to identify the role these corticosteroid receptors have in stress-related behavioral adaptation.

Rapid Down-Regulation of Glucocorticoid Receptor Gene Expression in the Dentate Gyrus after Acute Stress in vivo: Role of DNA Methylation and MicroRNA Activity.

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.

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

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.

A functional role for both -aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus.

Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K(+)-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased.

Insights into isoform-specific mineralocorticoid receptor action in the hippocampus.

The mineralocorticoid receptor (MR) plays a critical role in the mammalian brain as a mediator of appropriate cellular and behavioural responses under both baseline and stressful conditions. In the hippocampus, the MR has been implicated in several processes, such as neuronal maintenance, adult neurogenesis, inhibitory control of the hypothalamic-pituitary-adrenal axis, and learning and memory. Because of its high affinity for endogenous glucocorticoid hormones, the MR has long been postulated to mediate tonic actions in the brain, but more recent data have expanded on this view, indicating that the MR elicits dynamic responses as well. The complexity of the diverse molecular, cellular, and physiological functions fulfilled by the human, rat and mouse MR could at least partially be explained by the existence of different isoforms of the receptor. The structural and functional characteristics of these isoforms, however, have remained largely unexplored. The present article will review the current knowledge concerning human, rat, and mouse MR isoforms and evaluate seminal studies concerning the roles of the brain MR, with the intent to shed light on the function of its specific isoforms.

Genomic regulation of Krüppel-like-factor family members by corticosteroid receptors in the rat brain.

Hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) mediate glucocorticoid hormone (GC) action in the hippocampus. These receptors bind to glucocorticoid responsive elements (GREs) within target genes, eliciting transcriptional effects in response to stress and circadian variation. Until recently, little was known about the genome-wide targets of hippocampal MRs and GRs under physiological conditions. Following on from our genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus, we investigated the Krüppel-like factors (KLFs) as targets of MRs and GRs throughout the brain under circadian variation and after acute stress. In particular, Klf2, Klf9 and Klf15 are known to be stress and/or GC responsive and play a role in neurobiological processes including synaptic plasticity and neuronal differentiation. We found increased binding of MR and GR to GREs within Klf2, Klf9 and Klf15 in the hippocampus, amygdala, prefrontal cortex, and neocortex after acute stress and resulting from circadian variation, which was accompanied by upregulation of corresponding hnRNA and mRNA levels. Adrenalectomy abolished transcriptional upregulation of specific Klf genes. These results show that MRs and GRs regulate Klf gene expression throughout the brain following exposure to acute stress or in response to circadian variation, likely alongside other transcription factors.

Epigenetic mechanisms in stress and adaptation.

Epigenetic mechanisms are processes at the level of the chromatin that control the expression of genes but their role in neuro-immuno-endocrine communication is poorly understood. This review focuses on epigenetic modifications induced by a range of stressors, both physical and psychological, and examines how these variations can affect the biological activity of cells. It is clear that epigenetic modifications are critical in explaining how environmental factors, which have no effect on the DNA sequence, can have such profound, long-lasting influences on both physiology and behavior. A signaling pathway involving activation of MEK-ERK1/2, MSK1, and Elk-1 signaling molecules has been identified in the hippocampus which results in the phospho-acetylation of histone H3 and modification of gene expression including up-regulation of immediate early genes such as c-Fos. This pathway can be induced by a range of challenging experiences including forced swimming, Morris water maze learning, fear conditioning and exposure to the radial maze. Glucocorticoid (GC) hormones, released as part of the stress response and acting via glucocorticoid receptors (GRs), enhance signaling through the ERK1/2/MSK1-Elk-1 pathway and thereby increase the impact on epigenetic and gene expression mechanisms. The role of synergetic interactions between these pathways in adaptive responses to stress and learning and memory paradigms is discussed, in addition we speculate on their potential role in immune function.

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors.

Glucocorticoid hormones (GCs) - acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) - are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.

Unexpected effects of metyrapone on corticosteroid receptor interaction with the genome and subsequent gene transcription in the hippocampus of male rats.

Glucocorticoid hormones (GCs) play a pivotal role in many stress-related biological processes. In the hippocampus, GCs act through mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) to modify gene transcription. The involvement of GCs in biological processes has been investigated using the corticosterone (CORT)-synthesis blocker metyrapone. How metyrapone affects the action of GC at the genomic level still remains unclear. Therefore, we investigated the effects of this enzyme blocker on plasma CORT levels and hippocampal MR and GR binding to GC responsive elements (GREs) within the GC target genes Fkbp5 (FK506-binding protein 5), Per1 (Period 1) and Sgk1 (Serum- and glucocorticoid-activated kinase 1), as well as the transcriptional responses of these genes under control and acute stress conditions in rats. For comparison, we also investigated these endpoints in rats that had undergone adrenalectomy (ADX). Although metyrapone had no effect on baseline levels of CORT, the drug increased MR and GR to GRE binding within the GC target genes and the transcriptional activity of these genes. As expected, acute forced swim (FS) stress strongly increased plasma CORT levels, hippocampal MR and GR to GRE binding within Fkbp5, Per1 and Sgk1, and the transcriptional activity (mainly hnRNA levels) of these genes. Metyrapone attenuated, but did not abolish, these effects of stress on plasma CORT and MR and GR to GRE binding. The drug effects on FS-induced transcriptional activity were gene-dependent with a reduction seen in Fkbp5 hnRNA (but not Fkbp5 mRNA), an enhancement in Per1 hnRNA (but not Per1 mRNA), and no effect on both Sgk1 hnRNA and mRNA levels. ADX however completely abrogated the effects of FS on plasma CORT, as well as hippocampal MR and GR to GRE binding and transcriptional responses. Thus, in contrast to ADX, metyrapone produced inconsistent effects on GC-sensitive genomic endpoints that question its suitability as a tool in neuroendocrine and other research.

Corticosterone levels in the brain show a distinct ultradian rhythm but a delayed response to forced swim stress.

Circulating corticosterone levels show an ultradian rhythm resulting from the pulsatile release of glucocorticoid hormone by the adrenal cortex. Because the pattern of hormone availability to corticosteroid receptors is of functional significance, it is important to determine whether there is also a pulsatile pattern of corticosterone concentration within target tissues such as the brain. Furthermore, it is unclear whether measurements of plasma corticosterone levels accurately reflect corticosterone levels in the brain. Given that the hippocampus is a principal site of glucocorticoid action, we investigated in male rats hippocampal extracellular corticosterone concentrations under baseline and stress conditions using rapid-sampling in vivo microdialysis. We found that hippocampal extracellular corticosterone concentrations show a distinct circadian and ultradian rhythm. The PULSAR algorithm revealed that the pulse frequency of hippocampal corticosterone is 1.03 +/- 0.07 pulses/h between 0900 and 1500 h and is significantly higher between 1500 and 2100 h (1.31 +/- 0.05). The hippocampal corticosterone response to stress is stressor dependent but resumes a normal ultradian pattern rapidly after the termination of the stress response. Similar observations were made in the caudate putamen. Importantly, simultaneous measurements of plasma and hippocampal glucocorticoid levels showed that under stress conditions corticosterone in the brain peaks 20 min later than in plasma but clears concurrently, resulting in a smaller exposure of the brain to stress-induced hormone than would be predicted by plasma hormone concentrations. These data are the first to demonstrate that the ultradian rhythm of corticosterone is maintained over the blood-brain barrier and that tissue responses cannot be reliably predicted from the measurement of plasma corticosterone levels.

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.

The hippocampus is involved in learning and memory. Previously, we have shown that the acquisition of the behavioural immobility response after a forced swim experience is associated with chromatin modifications and transcriptional induction in dentate gyrus granule neurons. Given that both N-methyl-D-aspartate (NMDA) receptors and the extracellular signal-regulated kinases (ERK) 1/2 signalling pathway are involved in neuroplasticity processes underlying learning and memory, we investigated in rats and mice whether these signalling pathways regulate chromatin modifications and transcriptional events participating in the acquisition of the immobility response. We found that: (i) forced swimming evoked a transient increase in the number of phospho-acetylated histone H3-positive [P(Ser10)-Ac(Lys14)-H3(+)] neurons specifically in the middle and superficial aspects of the dentate gyrus granule cell layer; (ii) antagonism of NMDA receptors and inhibition of ERK1/2 signalling blocked forced swimming-induced histone H3 phospho-acetylation and the acquisition of the behavioural immobility response; (iii) double knockout (DKO) of the histone H3 kinase mitogen- and stress-activated kinases (MSK) 1/2 in mice completely abolished the forced swimming-induced increases in histone H3 phospho-acetylation and c-Fos induction in dentate granule neurons and the behavioural immobility response; (iv) blocking mineralocorticoid receptors, known not to be involved in behavioural immobility in the forced swim test, did not affect forced swimming-evoked histone H3 phospho-acetylation in dentate neurons; and (v) the pharmacological manipulations and gene deletions did not affect behaviour in the initial forced swim test. We conclude that the forced swimming-induced behavioural immobility response requires histone H3 phospho-acetylation and c-Fos induction in distinct dentate granule neurons through recruitment of the NMDA/ERK/MSK 1/2 pathway.

Water temperature determines neurochemical and behavioural responses to forced swim stress: an in vivo microdialysis and biotelemetry study in rats.

Forced swimming is a behavioural stress model increasingly used to investigate the neurocircuitry of stress responses. Although forced swim stress clearly is a psychological stressor (anxiety, panic), its physical aspects are often neglected. There are indications that behavioural and neurochemical responses to swim stress depend on the water temperature. Thus, we investigated the responsiveness of hippocampal serotonergic neurotransmission (important in the coordination of stress responses), and of behaviour and core body temperature to forced swimming at different water temperatures (19, 25 and 35 degrees C). In vivo microdialysis and biotelemetry in freely-behaving rats were used. Dialysates were analysed for serotonin (5-HT) and its metabolite 5-HIAA (5-hydroxyindoleacetic acid) by HPLC with electrochemical detection. Forced swimming in water at 25 and 19 degrees C decreased core body temperature by 8 and 12 degrees C, respectively. A rapid and pronounced increase in hippocampal 5-HT and 5-HIAA was found in rats that swam at 35 degrees C, whereas biphasic responses in 5-HT and 5-HIAA were observed at 25 and 19 degrees C. Also swim stress behaviour and post-stress home cage behaviour depended on the water temperature. Comparing the serotonergic and core body temperature changes revealed that a combination of two different 5-HT and 5-HIAA responses seems to shape the neurotransmitter response. Swimming-induced increases in hippocampal extracellular concentrations of 5-HT and 5-HIAA occurred at all water temperatures, but these increases were temporarily quenched, or concentrations were transistently decreased, when core body temperature fell below 31 degrees C in water at 25 or 19 degrees C. These data demonstrate that water temperature is a key factor determining the impact of forced swim stress on behaviour and neurochemistry, and underscore that changes in these parameters should be interpreted in the light of the autonomic responses induced by this stressor.

Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behavior and hormonal adaptation to stress.

Corticotropin-releasing hormone (CRH) is centrally involved in coordinating responses to a variety of stress-associated stimuli. Recent clinical data implicate CRH in the pathophysiology of human affective disorders. To differentiate the CNS pathways involving CRH and CRH receptor 1 (Crhr1) that modulate behavior from those that regulate neuroendocrine function, we generated a conditional knockout mouse line (Crhr1(loxP/loxP)Camk2a-cre) in which Crhr1 function is inactivated postnatally in anterior forebrain and limbic brain structures, but not in the pituitary. This leaves the hypothalamic-pituitary-adrenocortical (HPA) system intact. Crhr1(loxP/loxP)Camk2a-cre mutants showed reduced anxiety, and the basal activity of their HPA system was normal. In contrast to Crhr1 null mutants, conditional mutants were hypersensitive to stress corticotropin and corticosterone levels remained significantly elevated after stress. Our data clearly show that limbic Crhr1 modulates anxiety-related behavior and that this effect is independent of HPA system function. Furthermore, we provide evidence for a new role of limbic Crhr1 in neuroendocrine adaptation to stress.

Epigenetic mechanisms in stress-related memory formation.

Coping with stressful events is part of everyone's daily life. The organism's response to stress is a complex array of physiological and behavioral changes aimed at the preservation/protection of the organism during the stressful event as well as at stimulating adaptive and mnemonic processes in case the event would re-occur in the future. The hippocampus including its 'gate', the dentate gyrus, is highly involved in these processes. We have been collecting evidence suggesting that the transcriptional activation seen in dentate gyrus neurons, which are involved in the encoding of memories of a psychologically stressful event, requires chromatin remodeling in these neurons driven by the phosphorylation (at Serine10) and acetylation (at Lysine14) of histone H3. These particular epigenetic mechanisms are potentially of special interest for neuronal functioning as they are associated with the induction of hitherto silent genes. The phospho-acetylation of histone H3 is brought about by the concurrent activation of two, possibly converging, signaling pathways, being the glucocorticoid receptor and the NMDA/MAPK/ERK/MSK signaling pathways. Thus, we present a new model about how signaling to the chromatin can shape a specific gene transcriptional response in dentate granule neurons required for the encoding of memory of the stressful event.

Voluntary exercise impacts on the rat hypothalamic-pituitary-adrenocortical axis mainly at the adrenal level.

INTRODUCTION: Evidence is accumulating that the regular performance of exercise is beneficial for stress coping. However, the hypothalamic-pituitary-adrenocortical (HPA) axis of voluntarily exercising rats has never been comprehensively investigated. METHODS: Therefore, male Sprague-Dawley rats were given access to a running wheel in their home cage for 4 weeks in which they ran 4-7 km per night. RESULTS: After 4 weeks, the exercising animals showed significantly less body weight gain, less abdominal fat tissue, decreased thymus weight, and increased adrenal weight (relative to body weight). Furthermore, tyrosine hydroxylase (TH) mRNA levels were selectively increased in the right adrenal medulla indicating an increase in sympathoadrenomedullary capacity in exercising rats. No changes were observed in paraventricular corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP) and oxytocin mRNA levels. Mineralocorticoid receptor (MR) mRNA levels in hippocampus and glucocorticoid receptor (GR) mRNA levels in frontal cortex, parvocellular paraventricular nucleus and anterior pituitary were unchanged, whereas GR mRNA levels were increased in distinct hippocampal cell layers. Early morning baseline levels of plasma ACTH and corticosterone were similar in both groups. Interestingly, the response to different stressful stimuli (e.g. forced swimming, novelty) revealed that the exercising rats showed stressor-specific changes in HPA hormone responses. Forced swimming evoked a markedly enhanced response in corticosterone levels in the exercising rats. In contrast, if rats were exposed to a novel environment, exercising rats showed a much lower response in corticosterone than the control animals. However, the response in ACTH to either stressor was comparable between groups. Thus, in exercising rats physically demanding stressors evoke enhanced glucocorticoid responses whereas mild psychologically stressful stimuli such as novelty result in an attenuated glucocorticoid response. Interestingly, this attenuated hormone response corresponded with the observation that the exercising rats showed less anxious behaviour in the novelty situation. CONCLUSIONS: The differential responses in plasma corticosterone levels to different types of stress in the face of comparable responses in ACTH levels underscore the existence of critical regulatory control mechanisms at the level of the adrenal gland. We have hypothesized that changes in the sympathoadrenomedullary input may play an important role in these distinct glucocorticoid responses to stress. Our previous studies have shown similar changes in voluntarily exercising mice. Therefore, we conclude that the effects of exercise on the organism are not species-specific. Thus, our observations may have translational implications for the human situation.

Acute Stress Enhances Epigenetic Modifications But Does Not Affect the Constitutive Binding of pCREB to Immediate-Early Gene Promoters in the Rat Hippocampus.

The immediate early genes (IEGs) c-Fos and Egr-1 are rapidly and transiently induced in sparse neurons within the hippocampus after exposure to an acute stressor. The induction of these genes is a critical part of the molecular mechanisms underlying successful behavioral adaptation to stress. Our previous work has shown that transcriptional activation of c-Fos and Egr-1 in the hippocampus requires formation of a dual histone mark within their promoter regions, the phosphorylation of serine 10 and acetylation of lysine 9/14 of histone H3. In the present study, using chromatin immuno-precipitation (ChIP), we found that an increase in the formation of H3K9ac-S10p occurs within the c-Fos and Egr-1 promoters after FS stress in vivo and that these histone modifications were located to promoter regions containing cAMP Responsive Elements (CREs), but not in neighboring regions containing only Serum Responsive Elements (SREs). Surprisingly, however, subsequent ChIP analyses showed no changes in the binding of pCREB or CREB-binding protein (CBP) to the CREs after FS. In fact, pCREB binding to the c-Fos and Egr-1 promoters was already highly enriched under baseline conditions and did not increase further after stress. We suggest that constitutive pCREB binding may keep c-Fos and Egr-1 in a poised state for activation. Possibly, the formation of H3K9ac-S10p in the vicinity of CRE sites may participate in unblocking transcriptional elongation through recruitment of additional epigenetic factors.

Molecular and Epigenetic Mechanisms Underlying Cognitive and Adaptive Responses to Stress.

Consolidation of contextual memories after a stressful encounter is essential for the survival of an organism and in allowing a more appropriate response to be elicited should the perceived threat reoccur. Recent evidence has explored the complex role that epigenetic mechanisms play in the formation of such memories, and the underlying signaling pathways are becoming more apparent. The glucocorticoid receptor (GR) has been shown to play a key role in these events having both genomic and non-genomic actions in the brain. GR has been shown to interact with the extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) signaling pathway which, in concert, drives epigenetic modifications and chromatin remodeling, resulting in gene induction and memory consolidation. Evidence indicates that stressful events can have an effect on the offspring in utero, and that epigenetic marks altered early in life may persist into adulthood. A new and controversial area of research, however, suggests that epigenetic modifications could be inherited through the germline, a concept known as transgenerational epigenetics. This review explores the role that epigenetic processes play in the central nervous system, specifically in the consolidation of stress-induced memories, the concept of transgenerational epigenetic inheritance, and the potential role of epigenetics in revolutionizing the treatment of stress-related disorders through the emerging field of pharmacoepigenetics and personalized medical treatment.