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Epigenetic Regulation of Resistance to Emotional Stress: Possible Involvement of 5-HT1A Receptor–Mediated Histone Acetylation
J Pharmacol Sci 125, 347 – 354 (2014)
Journal of Pharmacological Sciences © The Japanese Pharmacological Society
Current Perspective
Epigenetic Regulation of Resistance to Emotional Stress: Possible Involvement of 5-HT1A Receptor–Mediated Histone Acetylation Minoru Tsuji1,*, Kazuya Miyagawa1, and Hiroshi Takeda1 Department of Pharmacology, School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara, Tochigi 324-8501, Japan
1
Received May 7, 2014; Accepted June 13, 2014
Abstract. The ability to resist stress is an important defensive function of a living body. Thus, elucidation of the mechanisms by which the brain resists stress could help to pave the way for new therapeutic strategies for stress-related psychiatric disorders including depression. The present review focuses on the roles of brain 5-HT1A receptor–mediated epigenetic mechanisms in the development of resistance to emotional stress. Behavioral pharmacological studies have demonstrated that treatment with a 5-HT1A receptor agonist 24 h before testing suppressed the decrease in emotional behaviors induced by acute restraint stress. Studies with DNA microarray technology have revealed that histone deacetylase genes were decreased in the hippocampus of mice that had been pretreated with a 5-HT1A receptor agonist 24 h beforehand. This preliminary finding was supported by data that hippocampal acetylated histone H3 was increased in mice that had developed emotional resistance to acute restraint stress by 5-HT1A receptor agonist. Furthermore, the histone deacetylase inhibitor trichostatin A also protected against the emotional changes induced by acute restraint stress, accompanied by the induction of histone H3 acetylation. These findings suggest that epigenetic mechanisms that are functionally coupled with 5-HT1A receptors may play a key role in the development of resistance to emotional stress. Keywords: 5-HT1A receptor, emotional stress resistance, epigenetic, histone acetylation
1. Introduction
various types of diseases, which are called stress-related disorders. This means that physiological systems activated by stress stimuli can not only protect and restore, but also damage a living body. Selye’s concept also forms the basis of research on neuropsychiatry. It has been speculated that the brain may have the ability to maintain a normal emotional state in response to stressors, but once this ability is disturbed by an uncontrollable stressor, the risk of the precipitation of stress-related psychiatric disorders including anxiety and depression may be increased. This speculation has led to a rich line of research on both the brain components and functions involved in resistance to stress events and dysfunction of this ability. It has been proposed that brain serotonin (5-HT) neuronal systems, especially 5-HT1A receptors, may be one of the primary mediators in the development of stress resistance (2). A large body of genetic epidemiological studies have shown that genetic factors participate in the onset of psychiatric disorders, but heritability varies according
Selye (1), who pioneered research on the biological effects of exposure to stress stimuli, described three stages in the response to external noxious stimulation: first, an initial brief alarm reaction associated with activation of the peripheral sympathetic-adrenomedullary system and the hypothalamic-pituitary-adrenocortical system; second, a prolonged period of resistance with adaptive responses that help to contain the stressful conditions; and third, a terminal stage of stress exhaustion and death characterized by a gradual decrease in stress resistance. Thus, while stress responses are by nature beneficial for protecting a living body, exaggeration and/or exhaustion of these responses can cause *Corresponding author. [email protected] Published online in J-STAGE on July 19, 2014 doi: 10.1254/jphs.14R07CP Invited article
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to the type of the disease. For example, the heritability of major depression, which is approximately between 31% and 42% (3), is considerably lower than that of other psychiatric disorders such as schizophrenia, which show heritability rates of 70% – 80% (4). These reports suggest that genetic factors alone do not fully account for the onset of psychiatric illnesses. Indeed, epidemiological evidence indicates that an increased risk for depressive disorders is associated with environmental factors, especially exposure to stressful life events (5). Thus, recent interest in research on stress-related psychiatric disorders has focused on the interplay between genetics and environmental factors. Especially, over the past decade, an increasing number of findings have suggested that epigenetics play an important role. The term ‘epigenetics’ refers to the potentially heritable, but environmentally modifiable, regulation of genetic function and expression that is mediated through non-DNA-encoded mechanisms, i.e., DNA methylation and chromatin/histone modifications (6). Recent human studies have provided mounting evidence that epigenetic mechanisms are involved in the pathophysiology of various types of psychiatric disorders: for example, postmortem studies of suicide victims with a history of depression and childhood abuse showed the differential expression of DNA methyltransferase (DNMT) subtypes (7) and promoter-wide hypermethylation of ribosomal RNA gene promoter (8), respectively. Taken together, the consequence of an intrinsically incorrect or inadequate adaptation process in response to detrimental environmental factors such as stress might also accompany the establishment of aberrant epigenetic signatures. In the present article, we will introduce recent preclinical findings which suggest that epigenetic mechanisms in the brain mediated by 5-HT1A receptors may play an important role in the development of emotional resistance to stressful events. 2. Brain 5-HT1A receptors and their emotional regulation The existence of various types of 5-HT receptors was first proposed by Bennett and Snyder in 1976 (9), and the application of molecular biological techniques enabled the subsequent discovery of many additional 5-HT receptor subtypes. There are now believed to be seven kinds of 5-HT receptor families, 5-HT1–7, that comprise a total of 14 structurally and pharmacologically distinct 5-HT receptor subtypes (10). Among them, 5-HT1A receptors are dense in the limbic brain areas, notably the hippocampus, lateral septum, amygdala, and cortical areas (projection sites of 5-HT neurons), and also in the dorsal and median raphe nuclei (cell-body sites of 5-HT
neurons). Studies that examined the effects of neuronal lesions on the abundance of 5-HT1A receptors have clarified that this receptor subtype is located both postsynaptic to 5-HT neurons in the projection site (post synaptic 5-HT1A receptors) and also on 5-HT neurons themselves as autoreceptors in cell-body sites (5-HT1A autoreceptors) (11). Since the activation of 5-HT1A receptors causes neuronal hyperpolarization (12), they have inhibitory effects on 5-HT neurons themselves as well as non-5-HT neurons. After the 5-HT1A receptor binding sites in the brain were identified, our understanding of the physiological functions of this receptor subtype quickly progressed. These advances were driven by the identification of selective 5-HT1A receptor agonists such as 2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene (8-OHDPAT) (13) and flesinoxan (14), and their application to clarify the pharmacological properties of 5-HT1A receptors. At present, 5-HT1A receptors are of particular interest in stress studies based on increasing preclinical and clinical evidence; for example, various types of 5-HT1A receptor agonists have shown promising results in both animal models and patients with stress-related affective disorders (15), and mice in which the 5-HT1A receptor function has been genetically modified have shown emotional abnormalities under stressful conditions (16). 3. 5-HT1A receptors contribute to the development of emotional resistance to stress stimuli The hypothesis that brain 5-HT1A receptors may play a role in the development of emotional resistance to stress stimuli is supported by our behavioral studies using the hole-board test (17, 18). The hole-board test is a simple method for measuring the response of an animal to an unfamiliar environment. Previously, the hole-board test has been used to assess emotionality and/or responses to stress in mice or rats (19). Some advantages of this test are that several behaviors can be readily observed and quantified, which makes it possible to comprehensively describe the animal’s behavior. To establish a more detailed behavioral analysis, we have developed an automatic hole-board apparatus (20). The automatic hole-board apparatus makes it possible to automatically measure changes in various exploratory activities of animals, and therefore, this system may be a useful tool for objectively estimating various emotional states of animals. Using the automatic hole-board apparatus, we examined the effects of 5-HT1A receptor agonists on the changes in the emotionality of mice produced by exposure to restraint stress. Such changes in the emotionality
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of mice were evaluated in terms of changes in exploratory activity, i.e., moving distance and number and duration of rearing and head-dipping behaviors, on the hole-board. In this study, 5-HT1A receptor agonists helped prevent various emotional changes produced by stress stimuli (18). Interestingly, pretreatment with the 5-HT1A receptor agonist flesinoxan or 8-OH-DPAT 24 h before exposure to stress suppressed the decrease in head-dipping behavior produced by acute restraint stress. We have previously found that typical benzodiazepine anxiogenics and anxiolytics as well as acute restraint stress have selective effects on head-dipping behavior in the holeboard test (20), indicating that this behavior may be sensitive to changes in the emotional state. Accordingly, these findings imply that the activation of 5-HT1A receptors may facilitate some mechanisms in a living body that are involved in the emotional resistance to stress stimuli. 4. Brain 5-HT1A receptors mediate epigenetic mechanism As mentioned above, we have obtained behavioral evidence that the administration of 5-HT1A receptor agonists induced the development of emotional resistance to stress stimuli 24-h later (18). Due to the delayed action of 5-HT1A receptor agonists, it is possible that changes in the expression of various genes may occur after 5-HT1A receptor activation. DNA microarray technology is a powerful method for determining which genes change in a tissue sample (21). Thus, we have tried to characterize changes in gene expression in the hippocampus of mice that had been pretreated 24 h beforehand with the 5-HT1A receptor agonist flesinoxan using DNA microarray technology. Preliminary behavioral experiments demonstrated that in the hole-board test, the decrease in head-dipping behavior of mice induced by restraint stress was reduced by pretreatment with flesinoxan 24 h beforehand, which confirmed the development of resistance to emotional stress. A DNA microarray analysis revealed 22 genes that showed more than 2-fold increase in flesinoxan-pretreated mice compared with control mice. These genes were involved in signal transduction, transcription, and translation, cell adhesion, transport, metabolism, and the immune response (Fig. 1A and Table 1). Furthermore, we also found 22 genes with more than 2-fold decrease in expression, which were associated with signal transduction, transcription, and translation, cell adhesion, transport, metabolism, cell cycle, and apoptosis (Fig. 1A and Table 2). Interestingly, the histone deacetylase (HDAC) 10 gene was one of these genes that showed decreased expression (Table 2), and some other types of histone
Fig. 1. Changes in gene expression in the hippocampus of mice that had been pretreated 24 h beforehand with the 5-HT1A receptor agonist flesinoxan. A) Correlation of log hybridization signal intensities for each of the approximately 34,000 transcripts on the mouse Gene Chip between saline- and flesinoxan-treated groups. Two-fold–increased and –decreased genes (22 of each) were found in the hippocampus of mice that had been pretreated with flesinoxan 24 h beforehand. Refer to Tables 1 and 2 for details. B) Changes in histone deacetylase (HDAC) family genes in saline- and flesinoxan-treated groups. Decreases in class II HDAC isoform (HDAC6, 9, and 10) genes were found in the hippocampus of mice that had been pretreated with flesinoxan 24 h beforehand, although change in HDAC6 gene was not quite statistically significant (P = 0.0542). *P < 0.05 vs. saline-treated group (Student’s t-test).
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M Tsuji et al Table 1. Genes with a differential expression between saline-treated group and flesinoxan-treated group of more than 2-fold increased, as determined by GeneChip analysis Fold change (ratio drug/saline)
Gene symbol
Gene name
Ebna1bp2 Slc25a2 Ugt1a2 Oaz3 Lhx6 Mgl1 Map3k12 4930415N18Rik Gp1bb Slc7a7 Uble1a Gja1 Ddx6 Eva Arl4 Rad54l Mlp Ms4a3 Gsto1 Kcna1 Snrpn Lif
EBNA1 binding protein 2 solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 2 UDP glycosyltransferase 1 family, polypeptide A6 ornithine decarboxylase antizyme 3 LIM homeobox protein 6 macrophage galactose N-acetyl-galactosamine specific lectin 1 mitogen activated protein kinase kinase kinase 12 hypothetical protein 5031404N07 glycoprotein Ib, beta polypeptide solute carrier family 7 (cationic amino acid transporter, y+ system), member 7 ubiquitin-like 1 (sentrin) activating enzyme E1A gap junction membrane channel protein alpha 1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 6 epithelial V-like antigen ADP-ribosylation factor-like 4 RAD54 like (S. cerevisiae) MARCKS-like protein membrane-spanning 4-domains, subfamily A, member 3 glutathione S-transferase omega 1 potassium voltage-gated channel, shaker-related subfamily, member 1 small nuclear ribonucleoprotein N leukemia inhibitory factor
deacetylase genes, especially class II HDAC isoforms (HDAC6 and 9), were also decreased, although change in the HDAC6 gene was not quite statistically significant (P = 0.0542) (Fig. 1B). HDACs are enzymes that remove acetyl groups from histone tails of the chromatin structure, and thus a decrease in these enzymes would promote histone acetylation. A genome-wide analysis demonstrated that histone acetylation is localized mainly within promoters of the genome (22). Therefore, it is speculated that brain 5-HT1A receptors may mediate epigenetic mechanisms, and the transcriptional activation induced by histone acetylation may be involved, at least in part, in the development of emotional resistance to stress stimuli. 5. Possible role of HDACs as prodepressive agents A number of studies provide the evidence that HDACs seems to be prodepressive agents. It has been recently reported that viral-mediated overexpression of hippocampal HDAC5 blocks the antidepressive-like effect of imipramine in socially defeated mice, and the downregulation of HDAC5 is observed in the hippocampus of socially defeated mice that had shown improvement in their depressive-like behavior by treatment with imipra-
2.004 2.008 2.020 2.033 2.049 2.079 2.079 2.088 2.183 2.208 2.257 2.268 2.273 2.315 2.370 2.433 2.469 2.513 2.584 2.632 3.115 6.944
mine (23). Other preclinical studies using the stress model have demonstrated that chronic variable stress produced the depressive-like behavior in mice, accompanied by increased HDAC5 expression as well as activity of sirtuin 1 (SIRT1), also known as one of the class III HDAC isoforms, in the hippocampus (24). Furthermore, in the peripheral leukocyte of depressive patients, HDAC2, 4, and 5 as well as SIRT1, 2 and 6 were increased only in the depressive but not in the remissive state, while HDAC6 and 8 were increased in all patients regardless of the state (25 – 27). These reports indicate that, although the critical isoform still remains unknown, HDACs may play a significant role in the pathophysio logy of stress-related psychiatric disorders including depression. 6. Association between resistance to emotional stress and histone acetylation induced by 5-HT1A receptor activation Based on preliminary findings in the DNA microarray study, we recently investigated the association between the development of emotional resistance to stress stimuli and histone acetylation induced by a 5-HT1A receptor agonist (28). A time-course analysis in the hole-board
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Table 2. Genes with a differential expression between saline-treated group and flesinoxan-treated group of more than 2-fold decreased, as determined by GeneChip analysis Fold change (ratio drug/saline)
Gene symbol
Gene name
Prrx1 Rapgef1 Lmyc1 Slc25a26 Ank2 Six5 Brms1 Wnt5b 4932411A10 Enpp6 Gosr1 AI846217 Racgap1 Ctso AI849976 Vat1 Ripk1 Rapgef3 Zfp98 Hdac10 Itgb4 Nr6a1
paired related homeobox 1 Rap guanine nucleotide exchange factor (GEF) 1 lung carcinoma myc related oncogene 1 solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 26 ankyrin 2, brain sine oculis-related homeobox 5 homolog (Drosophila) breast cancer metastasis-suppressor 1 wingless-related MMTV integration site 5B hypothetical protein 4932411A10 ectonucleotide pyrophosphatase/phosphodiesterase 6 golgi SNAP receptor complex member 1 hypothetical protein 9630005K15 Rac GTPase-activating protein 1 cathepsin O Ras-GTPase-activating protein SH3-domain binding protein vesicle amine transport protein 1 homolog (T californica) receptor (TNFRSF)-interacting serine-threonine kinase 1 Rap guanine nucleotide exchange factor (GEF) 3 zinc finger protein 98 histone deacetylase 10 integrin beta 4 nuclear receptor subfamily 6, group A, member 1
test showed that suppression of the decrease in headdipping behavior induced by acute restraint stress was observed at 24 h, but not at 0.5 or 48 h, after treatment with flesinoxan. This suggests that the development of emotional resistance to stress stimuli under flesinoxan may be a delayed-onset and reversible effect. A notable finding is that acetylated histone H3 (AcH3) was significantly increased in the hippocampus of mice that had been pretreated with flesinoxan 24 h before testing. On the other hand, no change in AcH3 was observed 0.5 or 48 h after pretreatment with flesinoxan. This temporal pattern of the change in AcH3 corresponds with that of the behavioral effects of flesinoxan in the hole-board test, in which suppression of the restraint stress–induced decrease in head-dipping behavior was only observed at 24 h after treatment. Taken together, our behavioral and biochemical findings suggest that the hyperacetylation of histone H3 that results from the stimulation of 5-HT1A receptors may be involved in the development of emotional resistance to stress stimuli (Fig. 2). In support of this hypothesis, a recent study using a rat model of individual differences in the response to stress revealed that low stress responder rats did not show depressivelike behavior after chronic social defeat together with an increase in histone H3 acetylation in the hippocampus,
0.497 0.494 0.486 0.481 0.459 0.424 0.418 0.418 0.403 0.395 0.387 0.384 0.377 0.364 0.353 0.353 0.350 0.349 0.346 0.334 0.247 0.240
indicating that hippocampal histone H3 acetylation may play an important role in the resistance to stress (29). 7. Direct evidence of the involvement of brain histone acetylation in the development of resistance to emotional stress HDAC inhibitors prevent histone deacetylation through the inactivation of HDACs, resulting in increased levels of histone acetylation. These drugs are powerful pharmacological tools for investigating epigenetic mechanisms in vivo. For example, the notion that histone acetylation is involved in depression is supported by studies showing that systemic or intracerebral administration of HDAC inhibitors produces an antidepressive-like effect (30 – 32). Thus, to more directly establish an association between histone acetylation and the development of emotional resistance to stress stimuli, we carried out additional studies using the HDAC inhibitor trichostatin A (28). Trichostatin A is a potent reversible HDAC inhibitor and thought to be approximately equally sensitive to all isoforms of HDACs (33). As a result, trichostatin A protected against the emotional changes induced by stress stimuli along with histone H3 acetylation. Although a drastic decrease in head-dipping behavior was
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Fig. 2. Schematic diagram of epigenetic changes related to the development of resistance to emotional stress. Activation of 5-HT1A receptors by treatment with the selective agonist flesinoxan suppressed the decrease in emotional behaviors of mice exposed to acute restraint stress and increased hippocampal acetylated histone H3. Similar behavioral and epigenetic changes were also induced by treatment with the histone deacetylase inhibitor trichostatin A. These findings suggest that epigenetic mechanisms, especially hippocampal histone H3 acetylation, may play a key role in the development of resistance to emotional stress.
observed immediately after exposure to acute restraint stress in the hole-board test, these behavioral changes were completely suppressed by trichostatin A, with a peak at 4 h after treatment. Importantly, the time-course of the increase in hippocampal AcH3 induced by trichostatin A is quite similar to that of the suppression of the acute restraint stress–induced decrease in head-dipping behavior in the hole-board test, i.e., a significant change was observed 2 h after treatment and peaked 4 h afterward. These results strongly suggest that the hyperacetylation of histone H3, which may induce euchromatin and active gene transcription, in the hippocampus may be a key epigenetic process in the development of emotional resistance to stress stimuli (Fig. 2). 8. What are the functional molecules related to the histone acetylation–mediated resistance to emotional stress? Although the distinct mechanisms that underlie the development of emotional resistance to stress stimuli that may be involved in the hyperacetylation of histone H3 are unclear, at least one hypothesis can be considered
based on previous reports. In our previous study, we found that the suppressive effect of pretreatment with 5-HT1A receptor agonists 24 h before testing on the restraint stress–induced decrease in head-dipping behavior in the hole-board test was blocked by co-pretreatment with metyrapone, an inhibitor of corticosterone synthesis, at doses that reduced only the increase in plasma corticosterone levels induced by 5-HT1A receptor agonists without changing the basal levels (18). These findings suggest that the HPA axis plays a significant role in the 5-HT1A receptor–mediated development of resistance to emotional stress. On the other hand, it has been reported that histone H3 acetylation is associated with regulation of the transcription of hippocampal glucocorticoid receptor (GR), a major modulator of HPA axis function (34). Therefore, it is possible that the hyperacetylation of histone H3 might positively affect the function of the HPA axis via GR transcription and lead to the development of emotional resistance to stress stimuli. Another functional molecule that may also play a role is brain-derived neurotropic factor (BDNF). A growing body of evidence indicates that BDNF plays a critical role in the pathophysiology as well as treatment of stress-
Epigenetics and Stress Resistance
related psychiatric disorders such as depression (35). Several reports have recently revealed that BDNF is a target molecule whose epigenetic regulation is implicated in depression models. For example, both antidepressants and electroconvulsive seizure, a well-known treatment for depression, induce histone H3 acetylation at promoter regions of the BDNF gene in the hippocampus of an animal model of depression that correlated with BDNF expression (23, 36). Furthermore, it has also been reported that BDNF expression in the hippocampus was enhanced in stress-resistant mice that overexpressed GR (37). These reports led us to speculate that histone H3 acetylation of the BDNF gene might be involved in the development of resistance to emotional stress. Further studies will be required to explore this hypothesis. 9. Conclusion The brain has physiological functions to maintain homeostasis by resisting stressful situations, but impairment of this stress resistance may promote various types of stress-related psychiatric disorders. Thus, studies of brain functions involved in the development of stress resistance have recently become an interesting subject with regard to the etiology of affective disorders such as anxiety and depression. In the present article, we proposed that brain 5-HT1A receptor–mediated epigenetic mechanisms, especially histone H3 acetylation, may play an important role in the development of emotional resistance to stress stimuli. However, we have still only observed global changes in histone acetylation, and the precise mechanisms, i.e., the specific functional molecules associated with 5-HT1A receptor–mediated histone H3 acetylation, that underlie stress resistance remain unknown. Further studies focused on this point may promote both our understanding of the etiology of affective disorders and the development of new therapeutic strategies. Acknowledgments This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI 22590086).
Conflicts of Interest The authors declare that they have no conflicts of interest to disclose.
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Journal of Pharmacological Sciences © The Japanese Pharmacological Society
Current Perspective
Epigenetic Regulation of Resistance to Emotional Stress: Possible Involvement of 5-HT1A Receptor–Mediated Histone Acetylation Minoru Tsuji1,*, Kazuya Miyagawa1, and Hiroshi Takeda1 Department of Pharmacology, School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara, Tochigi 324-8501, Japan
1
Received May 7, 2014; Accepted June 13, 2014
Abstract. The ability to resist stress is an important defensive function of a living body. Thus, elucidation of the mechanisms by which the brain resists stress could help to pave the way for new therapeutic strategies for stress-related psychiatric disorders including depression. The present review focuses on the roles of brain 5-HT1A receptor–mediated epigenetic mechanisms in the development of resistance to emotional stress. Behavioral pharmacological studies have demonstrated that treatment with a 5-HT1A receptor agonist 24 h before testing suppressed the decrease in emotional behaviors induced by acute restraint stress. Studies with DNA microarray technology have revealed that histone deacetylase genes were decreased in the hippocampus of mice that had been pretreated with a 5-HT1A receptor agonist 24 h beforehand. This preliminary finding was supported by data that hippocampal acetylated histone H3 was increased in mice that had developed emotional resistance to acute restraint stress by 5-HT1A receptor agonist. Furthermore, the histone deacetylase inhibitor trichostatin A also protected against the emotional changes induced by acute restraint stress, accompanied by the induction of histone H3 acetylation. These findings suggest that epigenetic mechanisms that are functionally coupled with 5-HT1A receptors may play a key role in the development of resistance to emotional stress. Keywords: 5-HT1A receptor, emotional stress resistance, epigenetic, histone acetylation
1. Introduction
various types of diseases, which are called stress-related disorders. This means that physiological systems activated by stress stimuli can not only protect and restore, but also damage a living body. Selye’s concept also forms the basis of research on neuropsychiatry. It has been speculated that the brain may have the ability to maintain a normal emotional state in response to stressors, but once this ability is disturbed by an uncontrollable stressor, the risk of the precipitation of stress-related psychiatric disorders including anxiety and depression may be increased. This speculation has led to a rich line of research on both the brain components and functions involved in resistance to stress events and dysfunction of this ability. It has been proposed that brain serotonin (5-HT) neuronal systems, especially 5-HT1A receptors, may be one of the primary mediators in the development of stress resistance (2). A large body of genetic epidemiological studies have shown that genetic factors participate in the onset of psychiatric disorders, but heritability varies according
Selye (1), who pioneered research on the biological effects of exposure to stress stimuli, described three stages in the response to external noxious stimulation: first, an initial brief alarm reaction associated with activation of the peripheral sympathetic-adrenomedullary system and the hypothalamic-pituitary-adrenocortical system; second, a prolonged period of resistance with adaptive responses that help to contain the stressful conditions; and third, a terminal stage of stress exhaustion and death characterized by a gradual decrease in stress resistance. Thus, while stress responses are by nature beneficial for protecting a living body, exaggeration and/or exhaustion of these responses can cause *Corresponding author. [email protected] Published online in J-STAGE on July 19, 2014 doi: 10.1254/jphs.14R07CP Invited article
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to the type of the disease. For example, the heritability of major depression, which is approximately between 31% and 42% (3), is considerably lower than that of other psychiatric disorders such as schizophrenia, which show heritability rates of 70% – 80% (4). These reports suggest that genetic factors alone do not fully account for the onset of psychiatric illnesses. Indeed, epidemiological evidence indicates that an increased risk for depressive disorders is associated with environmental factors, especially exposure to stressful life events (5). Thus, recent interest in research on stress-related psychiatric disorders has focused on the interplay between genetics and environmental factors. Especially, over the past decade, an increasing number of findings have suggested that epigenetics play an important role. The term ‘epigenetics’ refers to the potentially heritable, but environmentally modifiable, regulation of genetic function and expression that is mediated through non-DNA-encoded mechanisms, i.e., DNA methylation and chromatin/histone modifications (6). Recent human studies have provided mounting evidence that epigenetic mechanisms are involved in the pathophysiology of various types of psychiatric disorders: for example, postmortem studies of suicide victims with a history of depression and childhood abuse showed the differential expression of DNA methyltransferase (DNMT) subtypes (7) and promoter-wide hypermethylation of ribosomal RNA gene promoter (8), respectively. Taken together, the consequence of an intrinsically incorrect or inadequate adaptation process in response to detrimental environmental factors such as stress might also accompany the establishment of aberrant epigenetic signatures. In the present article, we will introduce recent preclinical findings which suggest that epigenetic mechanisms in the brain mediated by 5-HT1A receptors may play an important role in the development of emotional resistance to stressful events. 2. Brain 5-HT1A receptors and their emotional regulation The existence of various types of 5-HT receptors was first proposed by Bennett and Snyder in 1976 (9), and the application of molecular biological techniques enabled the subsequent discovery of many additional 5-HT receptor subtypes. There are now believed to be seven kinds of 5-HT receptor families, 5-HT1–7, that comprise a total of 14 structurally and pharmacologically distinct 5-HT receptor subtypes (10). Among them, 5-HT1A receptors are dense in the limbic brain areas, notably the hippocampus, lateral septum, amygdala, and cortical areas (projection sites of 5-HT neurons), and also in the dorsal and median raphe nuclei (cell-body sites of 5-HT
neurons). Studies that examined the effects of neuronal lesions on the abundance of 5-HT1A receptors have clarified that this receptor subtype is located both postsynaptic to 5-HT neurons in the projection site (post synaptic 5-HT1A receptors) and also on 5-HT neurons themselves as autoreceptors in cell-body sites (5-HT1A autoreceptors) (11). Since the activation of 5-HT1A receptors causes neuronal hyperpolarization (12), they have inhibitory effects on 5-HT neurons themselves as well as non-5-HT neurons. After the 5-HT1A receptor binding sites in the brain were identified, our understanding of the physiological functions of this receptor subtype quickly progressed. These advances were driven by the identification of selective 5-HT1A receptor agonists such as 2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene (8-OHDPAT) (13) and flesinoxan (14), and their application to clarify the pharmacological properties of 5-HT1A receptors. At present, 5-HT1A receptors are of particular interest in stress studies based on increasing preclinical and clinical evidence; for example, various types of 5-HT1A receptor agonists have shown promising results in both animal models and patients with stress-related affective disorders (15), and mice in which the 5-HT1A receptor function has been genetically modified have shown emotional abnormalities under stressful conditions (16). 3. 5-HT1A receptors contribute to the development of emotional resistance to stress stimuli The hypothesis that brain 5-HT1A receptors may play a role in the development of emotional resistance to stress stimuli is supported by our behavioral studies using the hole-board test (17, 18). The hole-board test is a simple method for measuring the response of an animal to an unfamiliar environment. Previously, the hole-board test has been used to assess emotionality and/or responses to stress in mice or rats (19). Some advantages of this test are that several behaviors can be readily observed and quantified, which makes it possible to comprehensively describe the animal’s behavior. To establish a more detailed behavioral analysis, we have developed an automatic hole-board apparatus (20). The automatic hole-board apparatus makes it possible to automatically measure changes in various exploratory activities of animals, and therefore, this system may be a useful tool for objectively estimating various emotional states of animals. Using the automatic hole-board apparatus, we examined the effects of 5-HT1A receptor agonists on the changes in the emotionality of mice produced by exposure to restraint stress. Such changes in the emotionality
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of mice were evaluated in terms of changes in exploratory activity, i.e., moving distance and number and duration of rearing and head-dipping behaviors, on the hole-board. In this study, 5-HT1A receptor agonists helped prevent various emotional changes produced by stress stimuli (18). Interestingly, pretreatment with the 5-HT1A receptor agonist flesinoxan or 8-OH-DPAT 24 h before exposure to stress suppressed the decrease in head-dipping behavior produced by acute restraint stress. We have previously found that typical benzodiazepine anxiogenics and anxiolytics as well as acute restraint stress have selective effects on head-dipping behavior in the holeboard test (20), indicating that this behavior may be sensitive to changes in the emotional state. Accordingly, these findings imply that the activation of 5-HT1A receptors may facilitate some mechanisms in a living body that are involved in the emotional resistance to stress stimuli. 4. Brain 5-HT1A receptors mediate epigenetic mechanism As mentioned above, we have obtained behavioral evidence that the administration of 5-HT1A receptor agonists induced the development of emotional resistance to stress stimuli 24-h later (18). Due to the delayed action of 5-HT1A receptor agonists, it is possible that changes in the expression of various genes may occur after 5-HT1A receptor activation. DNA microarray technology is a powerful method for determining which genes change in a tissue sample (21). Thus, we have tried to characterize changes in gene expression in the hippocampus of mice that had been pretreated 24 h beforehand with the 5-HT1A receptor agonist flesinoxan using DNA microarray technology. Preliminary behavioral experiments demonstrated that in the hole-board test, the decrease in head-dipping behavior of mice induced by restraint stress was reduced by pretreatment with flesinoxan 24 h beforehand, which confirmed the development of resistance to emotional stress. A DNA microarray analysis revealed 22 genes that showed more than 2-fold increase in flesinoxan-pretreated mice compared with control mice. These genes were involved in signal transduction, transcription, and translation, cell adhesion, transport, metabolism, and the immune response (Fig. 1A and Table 1). Furthermore, we also found 22 genes with more than 2-fold decrease in expression, which were associated with signal transduction, transcription, and translation, cell adhesion, transport, metabolism, cell cycle, and apoptosis (Fig. 1A and Table 2). Interestingly, the histone deacetylase (HDAC) 10 gene was one of these genes that showed decreased expression (Table 2), and some other types of histone
Fig. 1. Changes in gene expression in the hippocampus of mice that had been pretreated 24 h beforehand with the 5-HT1A receptor agonist flesinoxan. A) Correlation of log hybridization signal intensities for each of the approximately 34,000 transcripts on the mouse Gene Chip between saline- and flesinoxan-treated groups. Two-fold–increased and –decreased genes (22 of each) were found in the hippocampus of mice that had been pretreated with flesinoxan 24 h beforehand. Refer to Tables 1 and 2 for details. B) Changes in histone deacetylase (HDAC) family genes in saline- and flesinoxan-treated groups. Decreases in class II HDAC isoform (HDAC6, 9, and 10) genes were found in the hippocampus of mice that had been pretreated with flesinoxan 24 h beforehand, although change in HDAC6 gene was not quite statistically significant (P = 0.0542). *P < 0.05 vs. saline-treated group (Student’s t-test).
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M Tsuji et al Table 1. Genes with a differential expression between saline-treated group and flesinoxan-treated group of more than 2-fold increased, as determined by GeneChip analysis Fold change (ratio drug/saline)
Gene symbol
Gene name
Ebna1bp2 Slc25a2 Ugt1a2 Oaz3 Lhx6 Mgl1 Map3k12 4930415N18Rik Gp1bb Slc7a7 Uble1a Gja1 Ddx6 Eva Arl4 Rad54l Mlp Ms4a3 Gsto1 Kcna1 Snrpn Lif
EBNA1 binding protein 2 solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 2 UDP glycosyltransferase 1 family, polypeptide A6 ornithine decarboxylase antizyme 3 LIM homeobox protein 6 macrophage galactose N-acetyl-galactosamine specific lectin 1 mitogen activated protein kinase kinase kinase 12 hypothetical protein 5031404N07 glycoprotein Ib, beta polypeptide solute carrier family 7 (cationic amino acid transporter, y+ system), member 7 ubiquitin-like 1 (sentrin) activating enzyme E1A gap junction membrane channel protein alpha 1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 6 epithelial V-like antigen ADP-ribosylation factor-like 4 RAD54 like (S. cerevisiae) MARCKS-like protein membrane-spanning 4-domains, subfamily A, member 3 glutathione S-transferase omega 1 potassium voltage-gated channel, shaker-related subfamily, member 1 small nuclear ribonucleoprotein N leukemia inhibitory factor
deacetylase genes, especially class II HDAC isoforms (HDAC6 and 9), were also decreased, although change in the HDAC6 gene was not quite statistically significant (P = 0.0542) (Fig. 1B). HDACs are enzymes that remove acetyl groups from histone tails of the chromatin structure, and thus a decrease in these enzymes would promote histone acetylation. A genome-wide analysis demonstrated that histone acetylation is localized mainly within promoters of the genome (22). Therefore, it is speculated that brain 5-HT1A receptors may mediate epigenetic mechanisms, and the transcriptional activation induced by histone acetylation may be involved, at least in part, in the development of emotional resistance to stress stimuli. 5. Possible role of HDACs as prodepressive agents A number of studies provide the evidence that HDACs seems to be prodepressive agents. It has been recently reported that viral-mediated overexpression of hippocampal HDAC5 blocks the antidepressive-like effect of imipramine in socially defeated mice, and the downregulation of HDAC5 is observed in the hippocampus of socially defeated mice that had shown improvement in their depressive-like behavior by treatment with imipra-
2.004 2.008 2.020 2.033 2.049 2.079 2.079 2.088 2.183 2.208 2.257 2.268 2.273 2.315 2.370 2.433 2.469 2.513 2.584 2.632 3.115 6.944
mine (23). Other preclinical studies using the stress model have demonstrated that chronic variable stress produced the depressive-like behavior in mice, accompanied by increased HDAC5 expression as well as activity of sirtuin 1 (SIRT1), also known as one of the class III HDAC isoforms, in the hippocampus (24). Furthermore, in the peripheral leukocyte of depressive patients, HDAC2, 4, and 5 as well as SIRT1, 2 and 6 were increased only in the depressive but not in the remissive state, while HDAC6 and 8 were increased in all patients regardless of the state (25 – 27). These reports indicate that, although the critical isoform still remains unknown, HDACs may play a significant role in the pathophysio logy of stress-related psychiatric disorders including depression. 6. Association between resistance to emotional stress and histone acetylation induced by 5-HT1A receptor activation Based on preliminary findings in the DNA microarray study, we recently investigated the association between the development of emotional resistance to stress stimuli and histone acetylation induced by a 5-HT1A receptor agonist (28). A time-course analysis in the hole-board
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Table 2. Genes with a differential expression between saline-treated group and flesinoxan-treated group of more than 2-fold decreased, as determined by GeneChip analysis Fold change (ratio drug/saline)
Gene symbol
Gene name
Prrx1 Rapgef1 Lmyc1 Slc25a26 Ank2 Six5 Brms1 Wnt5b 4932411A10 Enpp6 Gosr1 AI846217 Racgap1 Ctso AI849976 Vat1 Ripk1 Rapgef3 Zfp98 Hdac10 Itgb4 Nr6a1
paired related homeobox 1 Rap guanine nucleotide exchange factor (GEF) 1 lung carcinoma myc related oncogene 1 solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 26 ankyrin 2, brain sine oculis-related homeobox 5 homolog (Drosophila) breast cancer metastasis-suppressor 1 wingless-related MMTV integration site 5B hypothetical protein 4932411A10 ectonucleotide pyrophosphatase/phosphodiesterase 6 golgi SNAP receptor complex member 1 hypothetical protein 9630005K15 Rac GTPase-activating protein 1 cathepsin O Ras-GTPase-activating protein SH3-domain binding protein vesicle amine transport protein 1 homolog (T californica) receptor (TNFRSF)-interacting serine-threonine kinase 1 Rap guanine nucleotide exchange factor (GEF) 3 zinc finger protein 98 histone deacetylase 10 integrin beta 4 nuclear receptor subfamily 6, group A, member 1
test showed that suppression of the decrease in headdipping behavior induced by acute restraint stress was observed at 24 h, but not at 0.5 or 48 h, after treatment with flesinoxan. This suggests that the development of emotional resistance to stress stimuli under flesinoxan may be a delayed-onset and reversible effect. A notable finding is that acetylated histone H3 (AcH3) was significantly increased in the hippocampus of mice that had been pretreated with flesinoxan 24 h before testing. On the other hand, no change in AcH3 was observed 0.5 or 48 h after pretreatment with flesinoxan. This temporal pattern of the change in AcH3 corresponds with that of the behavioral effects of flesinoxan in the hole-board test, in which suppression of the restraint stress–induced decrease in head-dipping behavior was only observed at 24 h after treatment. Taken together, our behavioral and biochemical findings suggest that the hyperacetylation of histone H3 that results from the stimulation of 5-HT1A receptors may be involved in the development of emotional resistance to stress stimuli (Fig. 2). In support of this hypothesis, a recent study using a rat model of individual differences in the response to stress revealed that low stress responder rats did not show depressivelike behavior after chronic social defeat together with an increase in histone H3 acetylation in the hippocampus,
0.497 0.494 0.486 0.481 0.459 0.424 0.418 0.418 0.403 0.395 0.387 0.384 0.377 0.364 0.353 0.353 0.350 0.349 0.346 0.334 0.247 0.240
indicating that hippocampal histone H3 acetylation may play an important role in the resistance to stress (29). 7. Direct evidence of the involvement of brain histone acetylation in the development of resistance to emotional stress HDAC inhibitors prevent histone deacetylation through the inactivation of HDACs, resulting in increased levels of histone acetylation. These drugs are powerful pharmacological tools for investigating epigenetic mechanisms in vivo. For example, the notion that histone acetylation is involved in depression is supported by studies showing that systemic or intracerebral administration of HDAC inhibitors produces an antidepressive-like effect (30 – 32). Thus, to more directly establish an association between histone acetylation and the development of emotional resistance to stress stimuli, we carried out additional studies using the HDAC inhibitor trichostatin A (28). Trichostatin A is a potent reversible HDAC inhibitor and thought to be approximately equally sensitive to all isoforms of HDACs (33). As a result, trichostatin A protected against the emotional changes induced by stress stimuli along with histone H3 acetylation. Although a drastic decrease in head-dipping behavior was
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Fig. 2. Schematic diagram of epigenetic changes related to the development of resistance to emotional stress. Activation of 5-HT1A receptors by treatment with the selective agonist flesinoxan suppressed the decrease in emotional behaviors of mice exposed to acute restraint stress and increased hippocampal acetylated histone H3. Similar behavioral and epigenetic changes were also induced by treatment with the histone deacetylase inhibitor trichostatin A. These findings suggest that epigenetic mechanisms, especially hippocampal histone H3 acetylation, may play a key role in the development of resistance to emotional stress.
observed immediately after exposure to acute restraint stress in the hole-board test, these behavioral changes were completely suppressed by trichostatin A, with a peak at 4 h after treatment. Importantly, the time-course of the increase in hippocampal AcH3 induced by trichostatin A is quite similar to that of the suppression of the acute restraint stress–induced decrease in head-dipping behavior in the hole-board test, i.e., a significant change was observed 2 h after treatment and peaked 4 h afterward. These results strongly suggest that the hyperacetylation of histone H3, which may induce euchromatin and active gene transcription, in the hippocampus may be a key epigenetic process in the development of emotional resistance to stress stimuli (Fig. 2). 8. What are the functional molecules related to the histone acetylation–mediated resistance to emotional stress? Although the distinct mechanisms that underlie the development of emotional resistance to stress stimuli that may be involved in the hyperacetylation of histone H3 are unclear, at least one hypothesis can be considered
based on previous reports. In our previous study, we found that the suppressive effect of pretreatment with 5-HT1A receptor agonists 24 h before testing on the restraint stress–induced decrease in head-dipping behavior in the hole-board test was blocked by co-pretreatment with metyrapone, an inhibitor of corticosterone synthesis, at doses that reduced only the increase in plasma corticosterone levels induced by 5-HT1A receptor agonists without changing the basal levels (18). These findings suggest that the HPA axis plays a significant role in the 5-HT1A receptor–mediated development of resistance to emotional stress. On the other hand, it has been reported that histone H3 acetylation is associated with regulation of the transcription of hippocampal glucocorticoid receptor (GR), a major modulator of HPA axis function (34). Therefore, it is possible that the hyperacetylation of histone H3 might positively affect the function of the HPA axis via GR transcription and lead to the development of emotional resistance to stress stimuli. Another functional molecule that may also play a role is brain-derived neurotropic factor (BDNF). A growing body of evidence indicates that BDNF plays a critical role in the pathophysiology as well as treatment of stress-
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related psychiatric disorders such as depression (35). Several reports have recently revealed that BDNF is a target molecule whose epigenetic regulation is implicated in depression models. For example, both antidepressants and electroconvulsive seizure, a well-known treatment for depression, induce histone H3 acetylation at promoter regions of the BDNF gene in the hippocampus of an animal model of depression that correlated with BDNF expression (23, 36). Furthermore, it has also been reported that BDNF expression in the hippocampus was enhanced in stress-resistant mice that overexpressed GR (37). These reports led us to speculate that histone H3 acetylation of the BDNF gene might be involved in the development of resistance to emotional stress. Further studies will be required to explore this hypothesis. 9. Conclusion The brain has physiological functions to maintain homeostasis by resisting stressful situations, but impairment of this stress resistance may promote various types of stress-related psychiatric disorders. Thus, studies of brain functions involved in the development of stress resistance have recently become an interesting subject with regard to the etiology of affective disorders such as anxiety and depression. In the present article, we proposed that brain 5-HT1A receptor–mediated epigenetic mechanisms, especially histone H3 acetylation, may play an important role in the development of emotional resistance to stress stimuli. However, we have still only observed global changes in histone acetylation, and the precise mechanisms, i.e., the specific functional molecules associated with 5-HT1A receptor–mediated histone H3 acetylation, that underlie stress resistance remain unknown. Further studies focused on this point may promote both our understanding of the etiology of affective disorders and the development of new therapeutic strategies. Acknowledgments This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI 22590086).
Conflicts of Interest The authors declare that they have no conflicts of interest to disclose.
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