Regulation of 5-HT Receptors and the Hypothalamic-Pituitary-Adrenal Axis

Disturbances in the serotonin (5-HT) system is the neurobiological abnormality most consistently associated with suicide. Hyperactivity of the hypothalmic-pituitary-adrenal (HPA) axis is also described in suicide victims. The HPA axis is the classical neuroendocrine system that responds to stress and whose final product, corticosteroids, targets components of the limbic system, particularly the hippocampus. We will review resulsts from animal studies that point to the possibility that many of the 5-HT receptor changes observed in suicide brains may be a result of, or may be worsened by, the HPA overactivity that may be present in some suicide victims. The results of these studies can be summarized as follows: (1) chronic unpredictable stress produces high corticosteroid levels in rats; (2) chronic stress also results in changes in specific 5-HT receptors (increases in cortical 5-HT2A and decreases in hipocampal 5-HT1A and 5-HT1B); (3) chronic antidepressant administration prevents many of the 5-HT receptor changes observed after stress; and (4) chronic antidepressant administration reverses the overactivity of the HPA axis. If indeed 5-HT receptors have a partial role in controlling affective states, then their modulation by corticosteroids provides a potential mechanism by which these hormones may regulate mood. These data may also provide a biological understanding of how stressful events may increase the risk for suicide in vulnerable individuals and may help us elucidate the neurobiological underpinnings of treatment resistance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73437/1/j.1749-6632.1997.tb52357.x.pd

Forced swim stress enhances the survival of new dentate gyrus neurons in rat through a glucocorticoid receptor-dependent mechanism

Adaptive and coping behaviours such as immobility in the forced swim (FS) retest are dependent on successful memory formation during initial training/tests. It has been suggested that stressful learning paradigms such as Morris water maze learning lead to enhanced survival of newly born neurons in the dentate gyrus (DG) and that these newly generated neurons may be involved in long-term memory formation. We have previously identified a signaling pathway, involving activation of ERK1/2 (extracellular signal-regulated kinase1/2), MSK1 (mitogen- and stress-activated kinase1), and Elk-1 (ets-domain- containing protein1) signaling molecules and activated within DG granule neurons following FS, which results in distinct epigenetic changes and induction of immediate early genes (IEGs; e.g. c-Fos/Egr-1) and the consolidation of the adaptive behavioural immobility response [1,2]. Glucocorticoid 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. We aimed to determine if this signaling pathway leading to IEG induction is activated specifically in young adult-born dentate neurons following FS. Rats were injected twice daily for 5 days with BrdU to label new neurons. One, six or twelve weeks after BrdU treatment the rats were forced to swim (15 min, 25°C-water), before being killed 1h later and tissue analyzed by immunofluorescence. FS-induced c-Fos was observed at all time points but there was no co-localization of the IEG with BrdU. Next, given that experiences during the first 1−2 weeks of a young adult-born DG neuron's life is important for its survival [3], we studied whether FS would promote neuronal survival. Rats were injected with BrdU for 5 days and split into groups: control and FS groups. The FS group underwent a 15-min FS challenge one week after the BrdU injections, whereas the control group remained in their home cages. Four weeks after the initial FS test both groups (i.e. control group and FS group) underwent another FS procedure and killed 1h later. The FS group, which had undergone a FS challenge four weeks previously, showed significantly more immobility behavior compared with controls (n = 8, p<0.01, Students t-test), indicating that they had remembered the initial FS test. No co-localization of c-Fos with BrdU was found in either group. The number of BrdU-positive neurons was higher in the dentate gyrus of the FS group (19.6±2.1 neurons, n = 8) than in control animals (12.0±1.1 neurons, n=4) (p<0.03, Student's t-test). To investigate a role of GRs in the survival-promoting effect of FS we pre-treated rats with the GR antagonist RU486 before the initial FS challenge. RU486 impaired the behavioural immobility response in the 4-week retest (n = 7/group; p<0.01, post-hoc Bonferroni test) and abolished the FS-evoked increase in neuronal survival compared with the vehicle-pre-treated group (n = 5/control group, 7/FS group; p<0.001, post-hoc Bonferroni test). Thus, a single FS challenge promotes the survival of new adult-born DG granule neurons, which requires GR activation at the time of the stressful challenge and which may contribute to the formation of long-term memories of the FS event

S-Adenosyl-methionine impairs forced swimming-induced behavioural immobility by inhibiting gene expression in dentate gyrus neurons

The consolidation of stress-induced adaptive behaviours, such as the learned immobility response in the forced swim (FS) test, depends on specific epigenetic modifications underlying gene transcriptional responses in dentate gyrus (DG) granule neurons of the hippocampus. In these neurons FS evokes the activation of two interacting signalling pathways, i.e. the glucocorticoid receptor (GR) and the NMDAR/ERK1/2/MSK1-Elk-1 pathways, resulting in phosphorylation of serine10 and acetylation of lysine14 at histone H3 (H3S10p-K14ac) which leads to induction of immediate early genes (IEGs) c-Fos and Egr-1 [1,2]. These molecular responses are critical for the consolidation of the behavioural immobility response [1,2].\ud \ud The drug and endogenous methyl-donor S-adenosylmethionine (SAM) impairs the consolidation of the behavioural immobility response [3] suggesting the involvement of histone methylation and/or DNA methylation in gene transcriptional control underlying the behavioural response but this is unknown. Therefore, to understand the mechanism of action of SAM and to gain insight into the involvement of histone/DNA methylation, rats were injected with SAM (100 mg/kg s.c.) 30 minutes before FS (15 min, 25ºC). Twenty-four hours later they were subjected to FS again and immobility behaviour were scored in 10 s bins. SAM had no effect on immobility in the initial FS test (mean±SEM; vehicle: 15.8±1.6 bins, n = 8; SAM: 15.0±1.9 bins, n = 9; P>0.05 post-hoc Bonferroni test). However, in the retest, immobility in the SAM group was significantly lower (11.4±3.3 bins, n = 9) than that in the vehicle group (20.6±2.3 bins, n = 8; P<0.05), confirming that SAM indeed impairs the consolidation of the behavioural immobility response [3]. \ud \ud To study whether SAM's effects on behaviour could be explained through effects on FS-induced epigenetic and transcriptional responses in DG neurons, rats were pre-treated with SAM or vehicle and submitted to FS or not (baseline control) and killed 1 h later. Compared to vehicle, SAM evoked a significant decrease in FSinduced c-Fos and Egr-1in DG neurons (Vehicle: c-Fos: 127.1±4.6 neurons n = 5, Egr-1: 18.4±2.5 n = 5; SAM: c-Fos 82.6±5.0 neurons n = 6, Egr-1 8.5±1.2 n = 5; both c-Fos and Egr-1: P<0.01). However, SAM had no effect on stress-induced H3S10p-K14ac in DG neurons suggesting that the drug effect was independent of this dual epigenetic mark. Next, we investigated the effect of SAM and FS on the methylation status of histone H3 lysine residues. Chromatin immuno-precipitation (ChIP) analysis revealed that SAM and FS had no significant effects on H3K4me3, H3K9me3 and H3K27me3 at the c-Fos and Egr-1 promoters. \ud \ud Recently, we have started investigating DNA methylation using bisulfite pyrosequencing and methyl-DNA IP (MeDIP) to assess the cytosine methylation status of CpG islands within the c-fos and egr-1 gene promoters. We found that cytosine methylation of both promoters was very low (<5%) in the DG, the rest of hippocampus, and the neocortex and did not change after FS. Presently we are S64 Epigenetics: towards new drug targets studying the effects of SAM on de novo DNA methylation of the gene promoters. \ud \ud We conclude that SAM impairs the FS-induced behavioural immobility response through inhibition of gene transcription in DG neurons. This drug effect appears to be independent of histone modifications

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

<br><strong><em>Background:</em></strong> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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 <i>Nr3c1</i> (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 <i>Nr3c1</i> gene. <b><i>Conclusions:</i></b> 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 <i>Nr3c1</i> gene, most likely facilitated by DNMT3a, and increased expression of miR-124a - could be responsible for these changes

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