Early stressful life events (ESLE) have been related to physical and mental health disorders in adults [1, 2]. These adverse events during childhood include interpersonal losses, violence, neglect, parental divorce and sexual abuse, among others ; domestic violence and parental divorce are the most common . The mental disorders associated with ESLE include depression, increased impulsivity and higher suicide risk. These disorders usually begin during childhood or adolescence, although many of them persist until adulthood [3-7]. Additionally, other clinical consequences such as early menarche and sexual risk behaviours in adolescence have been related to preschool anomalous maternal care, such as harshness .
From the psychological point of view, the high prevalence of psychiatric disorders has been related to the fact that adverse childhood events are associated with poorer mental representation of self-concept, for example ‘I'm not valuable’, and with arbitrary negative inferences, such as ‘I will be criticised’ [4, 9].
The prevalence of ESLE varies from one country to another, but it is estimated that at least half of the population has suffered one or more of these events in their early years. According to the World Health Organization (WHO) approximately 20% of women and 5–10% of men report being sexually abused in childhood, while 25–50% of all children report being physically abused . Also, many children have suffered emotional abuse (sometimes referred to as psychological abuse) and neglect.
Neuroanatomy and early-life stressful experiences
Consistent with previous clinical data, neuroanatomical studies have described that early stress is associated with alterations of some brain regions, such as the hippocampus and prefrontal cortex (PFC) among others. The hippocampus is involved in learning, memory and negative feedback of stress hormone secretions . It has connections with the amygdale and PFC, regions involved in emotions and cognition; thus alteration in these neurocircuits could account for the symptoms observed in mood and anxiety disorders . Reductions in the hippocampal volume have been observed in patients with major depressive disorder and post-traumatic stress disorder (PTSD), especially in adults with history of ESLE (12–14), and in personality disorders with history of child abuse . This hippocampal reduction is due to chronic stimulation of the hippocampal glucocorticoid receptors (GR) by circulating glucocorticoids (GC) . Chronic stress reduces hippocampal dendritic branching [17-19]. Similarly, chronic stress also reduces medial PFC dendritic branching [17-19]. In the amygdale the effect is opposite, with increased dendritic branching resulting in stimulus hyperreactivity [20, 21]. These changes may generate abnormal neurocircuitry, contributing to the development of mood and anxiety disorders. Also, a loss of the asymmetry of the hippocampus – the left side is normally larger than the right one – has been observed in individuals with PTSD . The size of the hippocampus correlates with the level of self-esteem and the internal locus of control, i.e. the ability to control events that occur to a person. Accordingly, the cortisol response to a stressor is lower at higher levels of self-esteem and internal locus of control . On the other hand, prolonged use of steroids has also been associated with hippocampal reduction . Finally, some studies have shown changes in total brain volume in patients with early trauma and PTSD; this effect is more pronounced in women .
Early stress effects on the HPA axis activity
The HPA axis displays a circadian rhythm that controls the secretion of adrenal corticoids. This system is activated by internal and external stimuli such as physical and psychological stressors . The activity of the HPA axis begins with the secretion of corticotropin-releasing hormone (CRH) from CRHergic neurons, located in the paraventricular nucleus (PVN) of the hypothalamus. Arginine vasopressin (AVP), also secreted by PVN, acts synergistically with CRH to stimulate the secretion of adrenocorticotropin (ACTH) from the pituitary [27, 28]. The activity of these neurons is regulated by excitatory afferents from the amygdale and inhibitory from the hippocampus . ACTH, in turn, stimulates the adrenal cortex to synthesise and secrete GC, the key mediator of the stress response. GC exerts its effects on a wide variety of physiological processes for adaptation to stress; activation of the sympathetic system, deactivation of the parasympathetic, adjustments of metabolism and regulation of the immune response. Finally, the activation of the HPA axis is normally counteracted by negative feedback regulation of the GC at the hypothalamic, pituitary and hippocampal levels [29, 30]. CRH, along with AVP, mediates the HPA axis regulation in response to acute and chronic stress. Alterations of normal HPA axis functioning have been observed in some mental disorders such as PTSD and depression, especially when there are histories of ESLE [31-34], with an altered synthesis and release of GC . Intriguingly, patients with PTSD exhibit alterations in the HPA axis, with both increased  and reduced cortisol levels compared to control groups . Also, depressed patients exhibit higher basal plasma levels of cortisol than healthy controls, most of whom normalise cortisol rhythm after antidepressant treatment . Accordingly, experiments of early exposure to stress carried out in rhesus monkeys showed augmented cortisol responses to mild stressors in adulthood . This effect is explained by a reduction in the hippocampal expression of GR . Moreover, studies performed with the dexamethasone (DEX)/CRH test, a sensitive evaluation test for HPA axis hyperactivity, showed increased responsiveness in depressed patients with a history of stressful life events compared to controls . In animal models, mineralocorticoid receptors (MR) and GR expression levels and functioning have been observed to be altered under stress , affecting neurobiology and behaviour. For instance, stress during early development in marmoset monkeys leads to long-term reduction in MR and GR levels, which is associated to stress-related behaviour in adults [42, 43]. The hippocampal volume reduction described above is mediated by the sustained GC levels and is associated with a reduction in adhesion molecules. This affects the functionality of the synapse and neural architecture, leading to neuronal loss and consequently to hippocampal atrophy . This evidence supports the central role of HPA axis dysregulation in developing some mental disorders and their relation with ESLE.
Epigenetic mechanisms of gene expression regulation
The mechanisms of epigenetic regulation are of particular interest in the study of interaction between genetic backgrounds and environmental conditions, as they provide the means by which the latter factor may affect gene expression. Epigenetics refers to heritable and functionally important modifications of chromatin that affect phenotypes without alterations in the DNA sequence . Epigenetic mechanisms include DNA methylation, histone modifications, chromatin remodelling and regulation by non-coding RNA (ncRNA), all of which influence chromatin structure and gene expression.
DNA methylation consists of the addition of a methyl group to position 5 of some cytosine nucleotides, predominantly at the CpG dinucleotides. This contributes to chromatin condensation and consequently to gene transcription inhibition . DNA methylation is a modification heritable through mitosis and stable over time, although they can change when cells differentiate or are under environmental influences. The enzymes responsible for catalysing the methylations are a family of DNA methyl-transferases (DNMT) [46, 47]. Conversely, no enzymes responsible for DNA demethylation have been described yet, although it is known that there is active demethylation . Once the DNA is methylated the chromatin silencing occurs by alteration of the structure of nucleosomes, interference by the methyl group in the binding of transcription factor or by recruiting proteins that bind methylated cytosines, such as methyl cytosine-binding domain proteins (MECP2, MBD1, MBD2, MBD3 and MBD4) and proteins of the Kaiso family . These proteins in turn recruit the enzymatic machinery necessary to establish silent chromatin, such as histone deacetylases .
On the other hand, the epigenetic regulation by histone modifications consists in differential covalent modifications of histones, including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation and biotinylation. Acetylation and methylation are the most studied . These changes are ‘written’ or ‘erased’ by histone-modifying enzymes, such as histone acetyltransferases (HAT), that acetylate lysine residues of histones; histone kinases that phosphorylate serine, threonine and tyrosine; and histone methyl transferases . Combinations of histone modifications and the positions of these modifications determine different accessibilities for transcription factors to the DNA and consequently define the gene expression profiles. This is currently known as the histone code hypothesis . The histone modifications participate in the gene expression regulation through two main mechanisms: by controlling the chromatin structure and therefore the accessibility to the DNA, and by the binding of effector molecules . For instance, it has been postulated that acetylation reduces the strength of the interaction between the DNA and the histones, through the reduction of positive charges of the lysines. This would loosen up chromatin conformation and therefore facilitates the access of the transcriptional machinery. On the other hand, many proteins that bind to chromatin by recognising modified histones have been identified, including proteins that promote chromatin condensation (HP1, heterochromatin protein 1) and chromatin remodelling complexes [50, 52].
These histone modifications occur in concert with DNA methylation either activating or repressing the chromatin (reviewed in 47). For instance, the methyl cytosine-binding protein, MeCP2, binds to methylated cytosines and in turn recruits HDACs that collaborate in the chromatin inactivation by histone deacetylation.
In the regulation of gene expression, different types of chromatin remodelling complexes collaborate by controlling the chromatin packaging in the eukaryotic cell nucleus and consequently the access to the DNA by regulatory proteins . The remodelling complexes are multiproteic structures that alter the conformation of chromatin by mobilisation, removal or assembly of nucleosomes [52, 54]. The chromatin conformation alteration promotes or interferes with the interaction between DNA and transcriptional regulatory factors and determines the conformation of active or silent chromatin states . For chromatin remodelling, both energy (ATP) and changes in the composition of the nucleosome are required. The chromatin remodelling also occurs in concert with histone modifications. For instance, bromodomain-containing proteins (such as Brg and the BAF180 subunit of SWI/SNF) are recruited by acetylated histone residues and may be involved in chromatin relaxation at these sites favouring transcriptional activation .
Finally, ncRNA represent a group of non-translated RNAs, including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs. They are involved in gene expression control at different levels, ranging from the DNA methylation, chromatin remodelling, control of the stability of other RNAs and processing and translation of the mRNA . For instance, the long non-coding RNAs (lncRNAs) trigger epigenetic modifications that maintain specific states of inheritable chromatin . These lncRNAs act by coating regions of the chromatin and recruiting remodelling complexes, which in turn promote some histone modifications that establish gene repression-specific patterns [57-59]. On the other hand, miRNAs  act by pairing with specific mRNA to whom they exhibit complementarity, and silence gene expression likely by induction of degradation or downregulating their translation . They participate in the regulation of gene expression of large networks and are very much involved in animal development, such as differentiation, maintenance and neuronal plasticity .
Epigenetic regulation in health and disease: histone modification and DNA methylation
As the epigenetic programming affects gene expression, epigenetic alterations may have significant consequences, affecting diverse human traits such as metabolic, immunological and behavioural traits . Furthermore, it has been shown that possession of genetic risk factors does not necessarily result in the development of a disorder, suggesting that environmental factors may also be involved . Therefore, something in addition to the DNA sequence such as epigenetic alterations may be responsible for the development of complex diseases, stressing the relevance of understanding the genome-environment interplay.
It has been described that some environmental effects on behaviour are mediated by epigenetic alterations [1, 2]. These effects could become more significant over a longer time span and greater frequency and magnitude of exposure to the external conditions [65-67]. Moreover, earlier life events can have long-lasting effects on epigenetic programming [68-70] and some epigenetic alterations can last potentially during the whole life span or, outstandingly, be transmitted across generations [63, 71-74].
During the last decade a growing number of articles exploring the relationship between ELSE, adult psychopathology and epigenetic alterations have been published [2-6, 8, 63, 68, 75-77]. Although DNA methylation is the epigenetic alteration most investigated, histone modification and miRNA expression have also been explored. For instance, in a model of maternal separation, a reduction in mRNA expression of the Histone Deacetylases (HDAC) 1, 3, 7, 8 and 10, accompanied by increased acetylation in lysine 12 of histone H4 in the adult and adolescent forebrain neocortex was observed . Interestingly, the reversion of H4K12 acetylation worsened the abnormal emotive phenotype elicited by ELSE. This phenotype is characterised by increased anxiety and depressive-like behaviours. Conversely, the potentiation of histone modification improves the antidepressant action. This implies that histone regulation seems to be an adaptive strategy that allows the individual to better deal with ELSE, specifically maternal separation.
Additionally, it has been described that the expression levels of a number of miRNA is altered after early stress exposition – maternal separation during 180 min a day between day 2 to 14 – in the medial prefrontal cortex. This alteration is dependant of the transcriptional regulator REST-4 . For instance, the miRNAs 132 and 212 (miR132 and miR212) expression levels were augmented immediately after the stressful situation (day 14) and also in adulthood. These two miRNAs, miR132 and miR212, are involved in the modulation of synaptic plasticity .
In the same way, changes in the patterns of DNA methylation have been described after exposure to early stressors, triggering HPA axis dysregulation and neurochemical and neuroanatomical alterations . Thus epigenetic alterations in genes related to these functions may be potentially involved in the susceptibility to develop diseases.
DNA methylation and dysregulation of the HPA axis
To reveal the mechanism involved in the HPA axis dysregulation, various animal models have been studied. For instance, in rhesus monkeys maternal abuse determines abusive parenting behaviour in the offspring. Strikingly, cross-fostering of infants between abusive and non-abusive mothers reverses the effects in the offspring, supporting that this behaviour is mostly determined by the early environment . Weaver observed similar behaviour in rats. He reported that high-maternal care behaviour during the early postnatal period, consisting of licking, grooming and arched back nursing (LG-ABN), is transmitted to the next generation . In contrast, the offspring of low-care mothers exhibited fearful behaviour, high reactivity of the HPA axis and reproduced the same maternal behaviour in adulthood. Again, when the offspring of low- and high-care mothers were cross-fostered, the offspring replicate the adoptive mother's behaviour. These results are consistent with environmentally rather than genetically controlled behaviour. Weaver also showed that this programming of behaviour is determined during the first week of life, and depends on GR gene epigenetic modifications [82, 83]. The differences in rat behaviour mentioned above were produced by early hypermethylation of the GR gene (>80%), specifically in the exon 17 GR promoter region of the offspring of low maternal care mothers. The hypermethylation reduces GR mRNA levels in the hippocampus, preventing the proper negative feedback of the HPA axis [75, 84]. Accordingly, higher and sustained levels of corticosterone were observed, explaining the persistence of the low maternal care behaviour in the next generation. This methylation alteration, subsequent to low care, has critical and lasting effects on the stress responsiveness of offspring . On the contrary, in the high caring rats the offspring exhibited neither fearful behaviour nor alterations in HPA axis reactivity, and the GR exon 17 was rarely methylated . DNA methylation attracts proteins that bind methylated cytosines and HDAC which deacetylate histones, helping to keep the DNA tightly packed . Trichostatin A inhibits the histone deacetylation, which in turn favour the DNA demethylation. The trichostatin A treatment of mature low-care animals resulted in higher GR expression and reduced HPA response to stress, supporting that hypermethylation is the intermediary between maternal care and stress reactivity . This is in accordance with the GR reduction observed in marmoset monkeys after early stress [42, 43]. A reduction in the binding of the nerve growth factor inducible (NGFI)-A transcription factor to the GR promoter along with GR gene hypermethylation was observed in low-care rats . In humans, few similar studies have been carried out. However, in a postmortem study, the methylation levels in the GR gene were higher in subjects who died by suicide than in those who died in accident. Moreover, higher rates of methylation were present in individuals with a history of abuse/neglect, which is consistent with the results observed in animal models . Conversely, no changes in the methylation levels of exon 1F, the human equivalent of the rat exon 17 GR promoter, were observed in a postmortem study carried out in subjects with history of depression but without history of abuse. However, a reduction in the transcript carrying the 1F first alternative exon was observed, which could be explained by a reduction in NGFI-A transcription factor levels in the hippocampus . This data also suggest that as well as methylation, the NGF1A transcription factor could be related to the epigenetic regulation of GR.
Other epigenetic alterations have been observed in genes involved in stress response, neurotransmission and neuroplasticity, including FK506-binding protein 5 (FKBP5), arginine vasopressin (AVP), oestrogen receptor alpha (ERα), 5-hydroxy-tryptamine transporter (5HTT) and brain-derived neurotrophic factor (BDNF).
The FK506-binding protein 5 (FKBP5) is a co-chaperone that modulates the activity of GC, making it especially important in stress-response regulation. The relevance of FKBP5 in the development of mental disorders including mood disorders and PTSD with child abuse has been acknowledged through several genetic association studies [89-91]. Indeed, FKBP5 genetic polymorphisms have been associated with suicide risk [92, 93], insecure-resistant attachment  and behavioural traits known as higher harm avoidance in women and less cooperativeness in men . Some of the genetic variations have been also related to altered sensitivity to cortisol and to FKBP5 protein expression, affecting the HPA axis response . Similarly, mice lacking the FKBP5 coding gene are less vulnerable to the adverse effects of 3 weeks of chronic social defeat stress, probably due to an increased GR sensitivity increasing the feedback regulation of HPA . On the contrary, mice exposed to high doses of corticosterone exhibited reduced methylation levels in two CpG islands of intron 1 of the FKBP5 gene in both hippocampus and hypothalamus, with elevation in FKBP5 expression . Thus, FKBP5 protein interferes with GR activity mediating corticosterone action over the feedback regulation of HPA. This data suggest that epigenetic regulation of this gene could be involved in the behavioural stress response.
Similarly, epigenetic regulation in response to early environmental influences of the arginine vasopressin gene – the modulator of CRH actions over HPA axis – has been described. An early-life stress model in mice (periodic infant-mother separation during early postnatal life) exhibited a reduction in the methylation levels of the AVP enhancer in the PVN . This is important, since ACTH secretion by the pituitary is synergistically regulated by the CRH and AVP neuropeptides [27, 28]. This reduction in methylation correlates with sustained AVP expression and augmented HPA axis activity, prompting the long-lasting endocrine and behavioural alterations observed until adulthood . In addition, early-life stress model in mice has shown behavioural alterations such as reduced stress-coping ability and memory deficits, and features frequently observed in depression and PTSD [99, 100]. Accordingly, treatment with SSR149415, antagonist of the AVPV1b receptor, reversed the mice's increased stress responses and impaired memory, indicating that the augmented AVP level is responsible, at least in part, for the early-life stress phenotype .
Oestrogens, through the coordination of the neuroendocrine system, exert diverse actions on sexual development, sexual behaviour and reproduction. In addition, in the brain they contribute to other functions such as learning, memory and emotions, and also have neurotrophic and neuroprotective properties. These oestrogen actions are mediated by two intracellular receptors; alpha (ERα) and beta (ERβ) . These receptors are expressed in different tissues including several brain regions. The ERα is expressed only in the ventromedial nucleus of the hypothalamus and subfornix, whereas ERβ is widely expressed in the brain. They are co-expressed in the arcuate nucleus and hippocampus, but in the latter the levels of ERβ are higher . In animal studies it has been shown that agonists of ERα are anxiogenic, while ERβ agonists are anxiolytic . On the other hand, it has been suggested that oestrogen may regulate the HPA axis through the control of CRH expression . The clinical importance of oestrogens in stress response has been evaluated in the study of differences in response to stress by gender. For instance, women in the follicular phase or women who use contraceptives have lower levels of free cortisol under stress compared with men or with women in the luteal phase [104, 105].
As previously described, rats exposed to low-care rearing reproduce the mother's conduct in terms of mothering, early menarche and sexual behaviour. At the molecular level, an increased methylation of the ERα gene in the low-care rats leads to a reduction in the oxytocin receptor levels. As a consequence during adulthood a reduction in responsiveness to oxytocin released in the postpartum has been described, altering mothering behaviour . This methylation is due to the low caring behaviours in the mother, expressed as less licking and arching back nursing for breast feeding .
5HTT mediates the reuptake of serotonin (5HT) into the presynaptic cell, terminating the action of this neurotransmitter. It is inhibited by various antidepressants with a consequent increase of 5HT in the synaptic cleft. Many associated studies have related this gene to several mental disorders including anxiety, depression and suicide risk, particularly in individuals with ESLE [107-112].
Accordingly, studies carried out in lymphoblast cells have shown epigenetic alterations in the 5HTT gene. Specifically, a CpG island located in an alternative first exon is regulated by methylation, generating reduced mRNA levels when it is methylated . Moreover, environmental trauma modulates the effects of genetic polymorphisms through alterations of methylation levels in the transporter. Thus in patients, high methylation levels have been related to the severity of unresolved trauma . In agreement, secure attachment ameliorates the effect of the genetic polymorphisms in relation to self regulation in human infants at 25, 38 and 52 months of age . Additionally, early exposure to maternal depression is related to alterations of epigenetic programming in the umbilical cord leukocytes of newborn infants and consequently affects HPA stress reactivity three months after birth [116, 117]. The latter evidence supports a transgenerational maternal effect in the epigenetic signatures, and supports the possibility of detection of epigenetic alterations in peripheral blood.
Neuroplasticity, BDNF and epigenetic alterations
The neurotrophin BDNF performs multiple functions in the central nervous system, from neuroplasticity to regulating mood, behaviour and stress response . BDNF has been associated with mental illnesses such as depression and anxiety disorder, among others [119, 120]. For instance, low serum BDNF levels have been observed in depressive patients without pharmacological treatment, which correlated with the severity of depression . Accordingly, antidepressant treatment increases serum BDNF levels of depressed patients . In animal models, the rise of corticoids induced by stress leads to a loss of cognitive abilities and to a reduction of the apical dendrites of pyramidal neurons of the CA3 region of the hippocampus, an effect related to reduction of BDNF expression [123-125]. Interestingly, BDNF knockdown mice in the dorsal dentate gyrus exhibit depressive-like behaviours  and BDNF overexpression in the hippocampus elevates the resilience to stress . BDNF, like other neurotrophic factors, increases neuronal survival through negative regulation of apoptotic cascades [128, 129]. These actions are exerted in part by regulation of the expression of BDNF itself .
Early stress (abuse and neglect) has been related to alterations in the BDNF gene methylation levels in animal models. For instance, rats exposed to abusive caregivers during the first 7 days of life (30 min daily) exhibited reductions in BDNF levels, with elevated methylation levels in PFC. Moreover, this change has been reported to be transmitted to the next generation [131, 132]. Also, in a fear, learning model changes in hippocampal pattern of methylation of the BDNF gene have been observed . This alteration correlates with different mRNA levels. This evidence supports the importance of the methylation of the BDNF gene in normal and altered functioning of this system. Similar results have been observed in response to environmental influences such as early social experiences, with reduced and long-lasting hippocampus and PFC mRNA and protein levels [131, 134-136].
Early environmental conditions configure the epigenetic state adapted to the context. For instance, differences in maternal care of LG-ABN rats depend on environmental circumstances. In the face of danger the low-caring mothers ‘prepare’ their kin to a harsh environment through epigenetic alterations, shaping their brain and other organs to achieve more adaptive behaviours such as defensive and reproductive strategies to survive. As a result of this adaptive situation, offspring tend to be more promiscuous, favouring larger progeny. Also, the offspring show less explorative behaviour and greater resistance to infections and starving . These features result in improved fitness under adverse conditions. By extension, it may be hypothesised that in humans the emotional architecture is an adaptation to the closer context that includes parents-infant attachment style, family group constitution, socioeconomic status, political conditions, etc. that interact with genetic predispositions. This genetic and environmental interaction programs a profile of adjusted responses to the demands of the environment. Consequently, impulsivity or an elevated startle reaction may be an adaptive strategy developed in a dangerous context such as a dysfunctional family (aggressive, abusive, violent, etc.). However, this behaviour could be maladaptive in a safe context where children do not feel in danger. Accordingly, indiscriminate attachment behaviour in foster care children could also be considered as an adaptive strategy, where children crave for protection . In biological terms, it can be hypothesised that this behaviour may be epigenetically controlled.
Epigenetic alteration is an excellent mechanism for controlling this behaviour, because it is a mechanism that could regulate gene expression and could be affected by the environment. Also it has been shown that it may be heritable across generations, although in absence of the stimuli the epigenetic alterations fade over generations .