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© Springer International Publishing AG 2017
R. Delgado-Morales (ed.), Neuroepigenomics in Aging and Disease, Advances
in Experimental Medicine and Biology, DOI 10.1007/978-3-319-53889-1_8
Anxiety and Epigenetics
Andrew A. Bartlett, Rumani Singh, and Richard G. Hunter
Abstract
Anxiety disorders are highly prevalent psychiatric disorders often comorbid with
depression and substance abuse. Twin studies have shown that anxiety disorders
are moderately heritable. Yet, genome-wide association studies (GWASs) have
failed to identify gene(s) significantly associated with diagnosis suggesting a
strong role for environmental factors and the epigenome. A number of anxiety
disorder subtypes are considered “stress related.” A large focus of research has
been on the epigenetic and anxiety-like behavioral consequences of stress.
Animal models of anxiety-related disorders have provided strong evidence for
the role of stress on the epigenetic control of the hypothalamic-pituitary-adrenal
(HPA) axis and of stress-responsive brain regions. Neuroepigenetics may con-
tinue to explain individual variation in susceptibility to environmental perturba-
tions and consequently anxious behavior. Behavioral and pharmacological
interventions aimed at targeting epigenetic marks associated with anxiety may
prove fruitful in developing treatments.
Keywords
Anxiety • Epigenetic • Stress • Glucocorticoid • Plasticity • Hippocampus
• Amygdala • Prefrontal cortex • Histone • DNA methylation • Noncoding RNA
A.A. Bartlett • R. Singh, Ph.D. • R.G. Hunter, Ph.D. (*)
Department of Psychology, University of Massachusetts,
100 Morrissey Blvd, Boston, MA 02125, USA
e-mail: Andrew.Bartlett001@umb.edu; Rumani.Singh@umb.edu; Richard.Hunter@umb.edu
8
146
8.1 Introduction
Anxiety disorders (ADs) are among the most common psychiatric disorders, occur-
ring in roughly a third of the US population. They are also highly comorbid with
depression and substance abuse disorders, and the pathogenesis of AD is likely
highly interrelated [1]. While anxiety disorders are heritable and genetic factors
play a role in anxiety disorders, most of the risk of these disorders is environmental
in nature [2]. Stress, particularly in early life, substance abuse, circadian, and micro-
biota have all been shown to have an influence on risk of anxiety disorders [3–6].
Further, it is likely that anxious phenotypes are influenced by more subtle factors
such as the interplay between an anxious parent and a child whose early life is
defined in part by adapting to that parent’s behavior [7, 8]. Indeed the latter case is
emblematic of one of the important distinctions between heritability, which can
include epigenetic mechanisms, both behavioral and molecular, and the strictly
genetic inheritance with which heritability, in general, is often conflated. ADs are
moderately heritable with most of the disorders in the classification showing herita-
bility in the range of 30% [9]. GWASs have generally not met the criterion of
genome-wide significance, and candidate gene approaches have also been relatively
unsuccessful [10]. However some genetic polymorphisms do show replicable asso-
ciations with AD, for example, the glucocorticoid receptor chaperone FKBP5 has
been associated with risk of post-traumatic stress disorder (PTSD) in individuals
with a history of child abuse in an African–American sample, and the same sample
also demonstrated a female-specific association with PTSD and the ADCYPAP1R1
receptor for the neuropeptide PACAP [11, 12]. The catechol-O-methyltransferase
(COMT) valine158methionine polymorphism has been repeatedly implicated in
risk of panic disorder, though with different alleles imparting risk in European ver-
sus Asian populations [13]. Even these findings point to the role of other contextual
factors like ancestry and sex as influences on the underlying genetics, developmen-
tal context also appears to influence the expression of genetic risk, as a study in a
Swedish cohort has shown that different risk factors act at different times across
adolescence and early adulthood [14].
While environmental factors like stress are clearly significant in many anxiety
disorders, their effects can vary wildly across individuals. The role of the environ-
ment is most clear with PTSD, where most individuals are resilient and only a frac-
tion go on to develop the disorder after a trauma exposure [15, 16]. The question of
differential susceptibility in AD is another to which genetic explanations thus far
fall short.
8.2 The Neuroanatomy of Anxiety Disorders
Human anxiety is defined by emotional symptoms as well as behavioral and physi-
ological phenotypes. Much of the work in understanding the underlying neuroanat-
omy involved in anxiety-related pathology has been done using animal models.
Specifically, the focus has been on conserved endocrine systems and brain regions
A.A. Bartlett et al.
147
that identify or respond to environmental threats. For example, noxious stimuli may
result in freezing behavior, sympathetic nervous system activation, and subsequent
endocrine response in both the rat and the human. Across species, the limbic system
and the prefrontal cortex appear to be crucial for regulating threat recognition and
response. The hormonal response to threat appears, likewise, remarkably similar
and feature highly conserved signaling pathways.
Within the limbic system are a number of structures necessary for threat response
and assessment. The amygdala, for instance, appears to be necessary for fear
responses. Patients with Urbach-Wiethe disease have compromised amygdala func-
tion and report loss of feelings of fear [17]. In rodents, fear conditioning models pair
a benign stimulus, the conditioned stimulus (CS), with a noxious stimulus, the
unconditioned stimulus (US). A frequently used example of a US is a foot shock
which elicits freezing behavior, an unconditioned response (UR). After pairing of
the CS with the US, the CS alone can elicit this freezing behavior. This freezing is
referred to as the conditioned response (CR). Lesioning the amygdala has been
shown to obliterate freezing behavior, the CR, in conditioned rats [18, 19].
Stimulation of the amygdala during CS presentation produces subsequent freezing
behavior to the CS without US pairing [20]. During encoding of fear memories,
hippocampal inputs to the amygdala appear to be necessary for CS-US pairing for
contextual clues [21]. Within the amygdala, various subnuclei have been shown to
regulate different processes. The central amygdala (CeA) appears to regulate CR
expression through projections to the periaqueductal gray (PAG) [22]. The lateral
amygdala (LA) appears to receive CS and US inputs through cortical and thalamic
innervations [23]. The stimulation of a subpopulation of neurons in the LA, when
paired with presentation of the CS, appears to be sufficient to generate a CR. The
basal amygdala (BA) appears to have a dual role in both CR expression and suppres-
sion [24]. Two distinct populations of neurons were identified in the BA, one inner-
vated by the hippocampus and the other innervated by the PFC [24, 25]. During
extinction of the CS-US pairing, the PFC appears to inhibit the BA and attenuate
freezing CR [26–28]. The pairing of the CS-US improves prediction of the US
allowing for rapid behavioral response. However, when the CS fails to correctly
predict the US, the association must not continue to persist else anxiety or avoid-
ance for the US has now generalized to benign stimuli. These regions are critical to
avoidant and anxiety-like behavior. The dysregulation of these circuits may lead to
recurrent avoidance or anxious behavior to inappropriate stimuli similar to the defi-
nition of human anxiety.
8.3 The Neuroendocrine Axis in Anxiety Disorders
The HPA axis is a critical component of the acute stress response. In response to a
stressor, the body must divert resources appropriately in order to efficiently address
the challenge at hand. In part, this tentative balance is achieved through the activa-
tion of the HPA axis. The HPA axis is a negative feedback loop that begins with the
release of arginine-vasopressin (AVP) and corticotrophin-releasing factor (CRF)
8 Anxiety and Epigenetics
148
into the pituitary portal from the paraventricular nucleus (PVN) in the hypothala-
mus. This release promotes the production of proopiomelanocortin (POMC) in the
pituitary. POMC is subsequently converted to adrenocorticotropic hormone (ACTH)
and released into the bloodstream. The adrenal gland produces corticosteroids in the
adrenal cortex as a consequence of ACTH. Corticosteroids are released into the
blood and bind to the mineralocorticoid (MR) and glucocorticoid receptor (GR). In
the PVN, pituitary and hippocampus GRs inhibit the production of CRF resulting in
negative feedback loop.
The adrenal gland also produces two other hormones, epinephrine and norepi-
nephrine, from the adrenal medulla in response to ACTH. These hormones do not
engage in a self-regulating negative feedback loop but are indirectly regulated
through the actions of GRs. The role of these hormones is to control the response of
the body and the peripheral nervous system, for instance, reducing digestion and
immune function while increasing heart rate and blood pressure acutely. Interestingly,
pharmacological interventions targeting norepinephrine receptors have proved
effective reducing phobias during fear memory reconsolidation [29]. These findings
suggest that the autonomic nervous system may remain a potential area for research
and intervention in stress-related anxiety disorders such as phobias and PTSD.
8.4 Epigenetic Factors
The relative prominence of the environment and the moderate contribution of
genetic factors to the pathogenesis of anxiety disorders have made the study of these
disorders through the lens of epigenetics a fruitful avenue of research in recent
years. Many molecular epigenetic mechanisms have now been implicated in AD,
including DNA and histone modification as well as noncoding RNA (ncRNA).
Epigenetics, in the strict molecular sense, refers to regulation of DNA sequences
that does not involve alteration of actual base composition. Transcription and other
genomic functions are regulated directly through epigenetic modifications that typi-
cally annotate DNA and its associated histones via acetylation, methylation, and
phosphorylation. These epigenetic marks are tightly linked to chromatin state as
complex of DNA, RNA, and protein. Open chromatin is associated with active tran-
scription, whereas closed chromatin is associated with transcriptional silencing.
Epigenetic marks that define the epigenotype include DNA methylation and various
modifications (e.g., methylation, acetylation) of histone proteins that are complexed
with DNA. DNA methylation occurs at cytosines of CpG dinucleotides and is cata-
lyzed by enzymes of the DNA methyltransferase family. DNA methylation may
inhibit gene expression by direct interaction with factors that repress transcription
or, indirectly, through recruitment of methyl-CpG binding proteins (MeCP2 and
MBDs) complexed with enzymes that modify histone proteins. These modifications
can transform chromatin from an active to a repressed state, or vice versa.
The role of the epigenome in etiology of anxiety disorders and variations in
behavior and neurological status can now be investigated. Of particular importance
in epigenetics research is the fact that epigenetic marks are modifiable both in the
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germ line and in somatic tissues by genetic, environmental, and stochastic factors.
Each cell in the human body possesses not only a genotype, identical in all somatic
cells of an organism, but also an epigenotype that is highly variable among the dif-
ferent tissues of an individual. Errors or alterations in epigenotype can occur as
primary stochastic events or secondarily in response to either genetic mutations (e.g.
transposition events) or environmental exposures. Therefore a discussion of poten-
tial epigenetic etiologies of anxiety disorders necessarily involves both genetic and
environmental factors. Dysregulation of genes that control epigenetic mechanisms
leads to a number of “epigenetic syndromes” falling into two groups. Those with
changes in genes regulating epigenetic marks include enzymes such as DNA meth-
yltransferases, methyl-binding proteins, and enzymes that affect histone modifica-
tion. The second category involves genes that are regulated by epigenetic marks, for
example, imprinted genes.
8.5 Epigenetics in Animal Models of Anxiety
Twin studies of generalized anxiety disorder have failed to identify either a genetic
basis for or strongly heritable component of the disorder [30]. This class of mental
health disorders is often comorbid with addiction [31]. Both involve pathological
behaviors that have a neurobiological basis. Over the last decade, increasing focus
has been placed on how gene-environment interactions mediated by epigenetic
molecular mechanisms might improve our understanding of the disease. Though
environmental influences including trauma and substance abuse are known con-
tributors to anxiety, it is difficult or impossible at present to examine molecular
epigenetic changes in the central nervous system of clinical populations, and given
the tissue-specific nature of epigenetic mechanisms, accessible peripheral tissues
such as blood or epithelial cells may not reflect the changes present in the brain. For
these reasons, animal models have been employed to mimic the signs of anxiety.
While symptoms, such as intrusive thoughts, are impossible to model in rodents or
nonhuman primates, sophisticated paradigms have been used to model aspects of
social anxiety, general anxiety, and more broadly anxious temperament. In rodent
models, common behavioral paradigms to assess anxiety-like behaviors include the
elevated plus maze (EPM), light/dark box (LD), open field test (OFT), social defeat
(SD), and the social interaction test (SIT). The EPM consists of two arms of open
platforms and two arms of closed platforms featuring three walls. The EPM is based
on an innate fear of heights and open spaces such that rodents prefer the closed
platforms to the open platforms. After quickly equilibrating to the testing arena, less
anxious rodents will explore and spend increasing time on the open platforms. The
LD box consists of two connected chambers, one illuminated while the other is not.
The natural preference of the rodent is the dark chamber; however, given time less
anxious rodents again will explore and spend increasing time in the light chamber.
The OFT is a square testing arena with four walls. In novel settings, rodents prefer
to remain unexposed to predators, in this case, close to the wall. After exposure, less
anxious animals will cross the arena exploring and spend increasingly more time in
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150
the center. SD paradigms vary to some extent but primarily involve repeated expo-
sure of a rodent to another dominating rodent. The exposed rodents display
depressive- like symptoms but also social avoidance. Social avoidance is most com-
monly measured using SIT. SIT is conducted in a two-chamber arena separated by
a wall to prevent contact but allow for other sensory exchanges, i.e., visual cues,
odor, and ultrasonic vocalizations. More anxious, socially avoidant rodents will
spend less time in the area closest to the neighboring chamber after habituation.
These consist of the major testing paradigms used to proximate anxiety in the rodent
and have allowed for a more comprehensive understanding of the neuroepigenetic
regulation of anxiolytic behavior.
Natural variation in susceptibility to clinical anxiety has been subject to increased
scrutiny in recent years. Early animal work suggested that gene-environment inter-
actions likely mediated anxiety outcomes as SD paradigms among other stressors
produced anxious phenotypes. Notably, an early study showed that susceptibility to
SD, as measured by reduced interaction time in the SIT, was correlated with DNA
methylation of CpG islands in the promoter of the CRH gene in paraventricular
nucleus (PVN) [32]. Natural variation in maternal care during the first week of life
was shown to differentially pattern the methylation of nr3c1 promoter of offspring,
modification that persisted into adulthood and corresponded to reduced glucocorti-
coid receptor expression and enhanced HPA axis activation to an acute stressor [33].
These offspring were later characterized as displaying differential anxiety-like
behaviors as a consequence of maternal care received as measured by the EPM and
OFT [34, 35]. In adult mice, voluntary exercise has been demonstrated to increase
nr3c1 expression while reducing miRNA-124, known to inhibit nr3c1, expression
[36]. Though in contradiction to other findings, voluntary exercise decreased time
in the open arms of the EPM suggesting an increasingly anxious phenotype.
Recently, long noncoding RNA (lncRNA) expression of gomafu in the prefrontal
cortex (PFC) has been shown to regulate time spent in the center of the OFT and
grooming time to suggest that expression of this lncRNA is necessary for reducing
anxiety-like behaviors [37]. Likewise, loss-of-function l3mbtl1, null mice show
reduced latency to enter the light chamber in the LD box and increased time spent
in the center of the OFT [38]. As l3mbtl1 codes for a methylated lysine domain
histone-binding protein, a so-called chromatin reader, this suggests that histone
lysine methylation is required for regulating anxiety-like behavior. In this vein, tlr4
null mice did not show increased synaptic enrichment of NR1 following in the short
term following repeated ethanol exposure nor increased GluR1 enrichment in the
long term in the mPFC compared to similarly treated wild-type controls. These tlr4
mice failed to show mPFC enrichment of acetylated-H4 at the promoter of fosB and
BDNF in response to ethanol exposure. This observation suggests that tlr4 is neces-
sary for histone H4 acetylation at fosB and BDNF following ethanol exposure and
appears to be necessary for ethanol-induced increases in anxiety-like behavior as
indicated by time spent in the open arms of the EPM [39]. In contrast, others have
shown that acute ethanol exposure reduces amygdalar miRNA-494 subsequently
increasing Cited2, CBP, and p300 expression. These changes were associated with
increased H3 acetylation in the central amygdala and anxiolysis [40].
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8.6 Transgenerational Epigenetics
Transgenerational epigenetic can be either direct inheritance of mRNAs, protein, or
DNA modification via the germline or indirect “inheritance” such that the feed-
forward phenotypic profile of the parent can lead to changes in either noncoding
RNA expression, histone modification, or DNA methylation. Indirect inheritance
was shown by Weaver et al. (2004) where cross-fostering experiments suggested
that maternal care alone determined GR 1-7 promoter methylation in offspring hip-
pocampi [33]. Morgan and Bale (2011), in a case of direct inheritance, showed that
prenatal stress can lead to alterations in stress sensitivity and miRNA expression in
the brains of male offspring [41]. These effects persist for several generations sug-
gesting direct inheritance of paternal miRNAs or DNA methylation via sperm.
Transgenerational effects have been consistently observed in the offspring of
Holocaust survivors [42–44]. Maternal PTSD of these survivors has been predictive
of offspring PTSD risk and increased corticosteroid sensitivity. In specific impor-
tance to this chapter, offspring of Holocaust survivors were found to be at a far
greater risk of developing an anxiety disorder compared to control, age-matched
offspring born to Jewish parents [42]. At this date, the number of generations out to
which this inheritance persists and affects offspring of survivors remains unknown.
Transgenerational non-genomic transmission of both maternal behavior and HPA
axis activation in rats was initially demonstrated by Meaney et al. [45]. The same
group showed that glucocorticoid sensitivity and anxiety-like behavior are patterned
by maternal care and can persist out for several generations [33, 35]. The level of
maternal care during the first week of life patterned the methylation of the GR 1-7
promoter and subsequently GR expression in the hippocampus. These phenotypes
can be reversed however by cross-fostering offspring of low-licking and grooming
dams with high-licking and grooming dams. In anxious adults of low-licking and
grooming dams, the phenotype can be reversed by supplication of an HDAC inhibi-
tor to the hippocampus [34, 35]. Conversely, in low-anxiety adults of high-licking
and grooming dams, the phenotype can be reversed by infusion of a methyl donor
to the hippocampus [35]. Interestingly, maternal care has also been shown to affect
peripheral oxytocin receptor (OXTR) methylation status in rats [46]. A recent clini-
cal study also found that peripheral OXTR methylation was associated with increased
frequency of anxiety and depression [47]. Genome-wide methylation analysis in
infants of mothers with depression and/or anxiety revealed a number of CpG islands
to be differentially methylated [48]. Similarly, increased methylation of the BDNF
gene in blood of adults has been linked to lower maternal care and interpersonal
violence-related PTSD [49, 50]. In addition, poor maternal care and anxiety has
been linked to risk of diabetes and metabolic syndrome in bonnet macaque off-
spring [51, 52]. In high- and low-anxiety bred rats, increased H3K9me3 accumula-
tion was found at both the GR and FGF2 promoters in the hippocampus [53]. This
group also found differences in DNA methylation of the FGF2 promoter in the
hippocampus between high- and low-anxiety rats. High-anxiety rats had reduced
DNA methylation and methyl-binding protein association at the FGF2 promoter,
which presumably was permissive for increased FGF2 expression [53]. This group
8 Anxiety and Epigenetics
152
also showed that FGF2 increases H3K9me3 association with both the GR promoter
and its own. This demonstrates a potential mechanism by which early-life perturba-
tions independent of maternal care can contribute to anxiety-like behavior across
generations.
8.7 Neuroepigenetic Effects of Early Stress on Anxious
Behaviors
Early-life stress has been demonstrated repeatedly to pattern stress reactivity and
anxious behavior. These changes persist beyond the time frame of the initial stressor
and often long into adulthood. The prenatal effects of stress lead to dysregulation of
the HPA axis associated mainly with changes GR expression [35]. Though these
findings were first reported in animal studies. Recently, these findings have been
recapitulated in longitudinal human studies. For instance, maternal prenatal anxiety
has been shown to predict internalizing and anxiety scores on the child behavior
checklist in the infant [49]. Further, differences in global DNA methylation were
observed at a number of CpG sites in neonatal cord blood of mothers affected by
anxiety during gestation [48]. Likewise, maternal PTSD has been shown to associ-
ate with both increased glucocorticoid sensitivity in the offspring of Holocaust sur-
vivors and increased offspring diagnosed with anxiety disorders [42]. Maternal
PTSD has also been demonstrated to be predictive of offspring PTSD and presum-
ably through inherited stress reactivity [43, 44]. These findings suggest that both the
prenatal environment and stress/trauma history may recruit epigenetic processes in
the intergenerational transmission of HPA axis dysregulation and anxiogenic conse-
quences. However, consideration of allostatic load must be of concern as severe and
mild stress have opposing roles on physiology and behavior. Allostatic load is the
cumulative effect of multiple stressors taking into consideration severity, duration,
and ability to cope with stressors [54, 55]. Consider the effects of a severe uncon-
trollable stressor, for example, maternal separation, on stress sensitivity in contrast
to a mild controllable stressor such as voluntary exercise. While maternal separation
sensitizes the HPA axis of the infant, voluntary exercise can promote resiliency to
future stressors [56, 57].
8.7.1 Prenatal Stress
In utero exposure to maternal stress and corticosteroids patterns the HPA axis of
infants ultimately altering synaptic connectivity, function, and behavioral responses
specifically those involved in stress adaptation [58–60]. Prenatal restraint stress has
been shown to impair offspring brain function and development reducing HPA axis
feedback and altering neuroplasticity [61]. Prenatal stress and glucocorticoid treat-
ment produce lasting behavioral changes such as spatial learning impairment and
increased anxiety-like behavior [58, 59]. In addition, mild stressors, for instance,
postnatal handling, have been shown to reduce these deficits as well as attenuate
A.A. Bartlett et al.
153
HPA axis sensitivity [58, 59]. Prenatal stress does so by altering synaptic connectiv-
ity, neurogenesis, and chromatin structure in stress-sensitive regions of the brain, for
example, in the PFC where offspring of maternally stressed dams show reduced
dendritic spine complexity and density [60]. Similarly, in both rodent and nonhu-
man primate models, prenatal stress retards hippocampal neurogenesis in the den-
tate gyrus. Prenatal stress has been linked to increased methylation of the GR 1-7
promoter in the hippocampus as well as reduced methylation of the CRF promoter
in the hypothalamus and amygdala of male but not female mice [62]. These sex-
specific changes have been linked to differential expression of DNA methyltransfer-
ase 1 (DNMT1), though the changes responsible for this dichotomized expression
remain unknown. Elliot et al. (2010) first ascribed natural variation in social interac-
tion following social defeat in adults to be due in part to the methylation of the
CRH promoter in the hypothalamus. Mice susceptible to social defeat show
increased social anxiety and reduced CRH promoter methylation in the PVN [32].
The methylation status of the CRF promoter in PVN helps to explain natural vari-
ability in the susceptibility of mice to social defeat and consequently social anxiety.
Prenatal stress had previously been shown to differentially affect CRF release in the
PVN [63]. Interestingly, a subsequent study found that prenatal restraint stress
increased both anxious behavior and corticosterone release in response to stress
while reducing CRF promoter methylation at the same CpG islands noted by Elliot
et al. (2010) [64]. Prenatal restraint stress has also been shown to increase methyla-
tion of the REELIN promoter in the PFC perhaps linking changes in synaptic con-
nectivity observed there to underlying molecular influences [65]. REELIN is an
important neuroplasticity gene, known to be epigenetically regulated by fear condi-
tioning [66]. Similarly, prenatal exposure to maternal depression and anxiety has
been linked to increased NR3C1 1F promoter methylation and increased salivary
cortisol following exposure to a stressor in infants [67, 68]. Maternal anxiety has
been linked to differential methylation of a number of other genes in cord blood
including IGF2 and H19 [69]. In fact, distress during pregnancy has been linked to
placental methylation of a number of stress-related genes including HSD11B2,
NR3C1, and FKBP5 [70]. Other perturbations, including maternal diet and paternal
exposure to drugs of abuse such as cocaine and ethanol, have been shown to alter
cortical gene expression through changes in the epigenetic machinery and affect
anxiolytic behavior in the offspring [71–73]. Importantly, mild postnatal stressors
have been shown to reverse the effects of prenatal stress as well as promote resil-
iency [58, 59, 74, 75]. Given the association between maternal stress and anxiety,
these findings provide evidence for the efficacy of behavioral therapy and alike as
an early-life intervention [7, 76].
8.7.2 Early-Life Stress
The vast majority of studies of early-life stress focus on the epigenetic consequences
of the interactions within the mother-infant dyad. Both maternal care and separation
have been demonstrated to both alter HPA axis stress reactivity and adult anxiety
8 Anxiety and Epigenetics
154
behaviors of the infant through lasting changes to the epigenomes [35, 77].
Specifically, maternal separation has been shown to sensitize offspring HPA axis
activation early-life interventions including environmental enrichment attenuate
this effect [78]. Poor rearing conditions have been shown to increase CRF release
from the PVN and amygdala as well as hypermethylate the GR 1-7 promoter in the
hippocampus [33, 79]. Conversely, good maternal care and rearing conditions have
been demonstrated to hypomethylate the GR 1-7 promoter in the hippocampus,
produce efficient stress responses, and reduce anxiety-like behaviors [35, 80–82].
The GR 1F promoter is the human ortholog of the rodent 1-7 promoter [83].
Hypermethylation of the 1F promoter in the brains of suicide victims was associated
with childhood abuse [84]. The findings of McGowan et al. (2009) were later
expanded to include the 1-B, 1-C, and 1-H promoters as well [85]. Other groups
have failed to replicate some of these findings, however [86]. The McGowan group
has also shown that hippocampal ribosomal RNA expression is reduced in suicide
victims suggesting reduced hippocampal protein synthesis [87]. Childhood adver-
sity has also been linked to increased 1F promoter methylation in peripheral cells as
well [88, 89]. Methylation patterns as a consequence of childhood abuse over-
whelmingly persist into adulthood [90]. Early postnatal stress followed by subse-
quent adult chronic stress has been linked to reduced hippocampal plasticity and
increased anxiety-like behaviors [91]. Maternal separation has been shown to reduce
amygdalar neurotensin receptor 1 (NTSR1) expression through increased methyla-
tion of the NTSR1 promoter. Microinfusion of NTSR1 receptor agonist increased
conditioned freezing responses, while an agonist reduced this behavior suggesting
an epigenetic molecular mechanism sufficient for increasing anxiety-like behavior
[92]. Similarly, maternal separation has been linked to increased HPA activation to
environmental stressors in adult offspring [93]. More recently, however, this finding
was both replicated and associated with hypomethylation of the POMC, the gene
encoding the precursor for ACTH, in the pituitary [94]. As HPA axis dysregulation
has been associated with anxiety-like outcomes, again these findings suggest a criti-
cal role of these molecular influences as a consequence of stress in the context of
anxiety outcomes. Clinical work has recently shown that early childhood trauma
affects CpG methylation in both the promoter and gene proper of the 5-HT3Ar in
blood [95]. Interestingly, this locus is downstream of GR response element which
showed altered CpG methylation associated with emotional neglect and CpG meth-
ylation associated with anxiety-related behaviors.
Adolescence represents another postnatal life stage sensitive to the epigenetic
effects of stress [96–100]. For instance, chronic variable stress during adolescence
reduces hippocampal volume and spatial cognition, these effects persisting into
adulthood [101]. Isolation rearing in adolescent mice reduces the expression of
5-α-reductase I, the rate-limiting enzyme for allopregnanolone, a hormone shown
to reduce depressive- and anxiety-like symptoms in rodents [102, 103]. Isolated
juveniles show increased CpG methylation upstream of the transcription start site
of the SRD5A1 gene, which codes for this enzyme; one of these islands was dem-
onstrated to be sufficient to reduce expression in the PFC [102]. In adolescent
rhesus monkeys, anxious temperament is associated with increased methylation
A.A. Bartlett et al.
155
and reduced expression of the BCL11A and JAG1 genes, associated with neuro-
plasticity, in the amygdala [104]. Similarly, these findings have been supported by
recent clinical work identifying a correlation between NR3C1, the gene coding for
the glucocorticoid receptor, 1F promoter methylation in blood, and internalizing
symptoms [105]. Moreover, these adolescents showing increased 1F promoter
methylation and displaying internalizing behavior also had higher concentrations
of cortisol upon waking. These findings, in tandem, indicate a significant role of
neuroplasticity and HPA axis regulation in stress-sensitive regions of the brain,
notably the hippocampus, amygdala, PVN, and PFC, during adolescence and may
underscore potential individual variations that contribute to anxious susceptibility.
These epigenetic predispositions may be compounded by other environmental per-
turbations such as exposure to drugs of abuse. Intermittent alcohol exposure, for
instance, has been shown to increase HDAC activity in the rodent amygdala [106].
These changes were also associated with reduced time spent in the open arms of
the EPM and in the light compartment of the dark/light box into adulthood. Further
alcohol-exposed adults had reductions in the number of spines and increased alco-
hol intake. Conversely, acute alcohol exposure during adolescence produces simi-
lar changes in anxiety-like behaviors while decreasing HDAC activity in the rodent
amygdala [107]. In summation both predisposition and environmental perturbation
may work in synchrony during adolescence to dysregulate both transcription and
synaptic integrity in the amygdala and ultimately help shape entrain anxious
behavior.
8.8 Stress in Adulthood
Stress induces lasting changes in heterochromatin structure, ultimately changing
neuronal plasticity and behavior. The hippocampus, PFC, and amygdala are targets
of glucocorticoids. As these regions help regulate spatial memory, executive func-
tion, and fear responses, respectively, they are of the utmost importance in the con-
text of anxiety. These regions are extremely sensitive to both acute and chronic
stressors and express a large number of epigenetic enzymes and display profound
structural changes at the synaptic level in response to environmental stressors.
Stressors often produce some type of learning, the spatial and contextual compo-
nents of which are presumed to be coded by the hippocampus and the cue-based
components coded by the amygdalar [24, 108]. The reconsolidation and extinction
of these associations are mediated by the PFC. Dysregulation of these memories
may fail to attenuate improper responses to environmental stimuli, much like the
symptoms of anxiety. Fear conditioning is widely used to study learning and neuro-
plastic consequences thereof as well as to model symptoms of a number of anxiety
disorders as well as other stress-related disorders such as post-traumatic stress dis-
order [109, 110]. Epigenetics has been thought to be a potential basis of memory on
the molecular level [111–113]. Initially, Sweatt et al. (2004) first demonstrated the
role of hippocampal histone acetylation during fear memory formation [114]. Miller
and Sweatt (2007) later showed that fear conditioning upregulated expression of
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hippocampal DNMT3A and 3B, and that DNMT activity there was required for fear
memory consolidation [66]. Hippocampal methylation of reelin, PP1, and BDNF
was also changed by fear conditioning [115]. Interestingly, reelin and BDNF have
well-established roles in dendritic remodeling, and PP1 codes for a phosphatase that
acts at histone H3S10 [116, 117]. Presumably these are the grounds for its role in
memory as the dual acetylation-phosphorylation H3 mark was enriched at the BDNF
locus in the hippocampus. Others have found similarly that both histone modifica-
tion and DNA methylation play critical roles in the amygdala in memory reconsoli-
dation and consolidation, respectively [118]. Tsai et al. (2007) established that both
environmental enrichment and HDAC inhibition were sufficient for restoring defi-
cits in memory and synaptic connectivity in a mouse model of neuronal cell loss
[119]. A later study by the same group (2009) identified HDAC2 to be necessary for
the negative impacts on memory [120]. Recall of recent memories results HDAC2
dissociation from the chromatin, which causes increases in H3 acetylation and
increased expression of immediate early genes [121]. Recall of less recent memo-
ries do not produce such profound changes in HDAC activity. Yet, HDAC inhibition
during reconsolidation of remote fear memories allows for H3 acetylation, increased
immediate early gene expression, and neuroplastic changes [121]. This suggests
that epigenetic control of chromatin structure regulates neuroplastic changes under-
pinning behavioral outputs related to fear memories.
Social defeat represents another type of stress-based learning producing an anx-
ious phenotype in the defeated. Social defeat is a well-characterized animal model
of a number of psychiatric disorders including modeling symptoms of depression
and anxiety [122]. The Nestler group was early in demonstrating that social defeat
affects hippocampal chromatin signatures [123]. They showed that chronic social
defeat increased H3K27me3 repression of the BDNF promoter in the hippocampus.
Also, the accumulation of this repressive mark was mitigated by antidepressant
treatment, inhibiting HDAC2, resulting in increases in H3 acetylation and H3K4
methylation, both marks promoting transcription [123, 124]. The same group also
showed that chronic stress or cocaine exposure altered HDAC activity in the nucleus
accumbens [125]. DNMT3A expression increases as a consequence of chronic
defeat and decreases as a consequence of chronic cocaine which were associated
with synaptic changes as well in the same nuclei [126]. Interestingly, natural varia-
tion to susceptibility to social defeat has been associated with distinct methylation
signatures of the CRF promoter in the PVN [32]. Resilient animals also show
increased H3K9me3 and K3K27me3 in the nucleus accumbens [127, 128]. The
levels of accumbal H3K9me3 also change in response to cocaine exposure as well
as dendritic morphology [129]. Acute stress and chronic antidepressant treatment
have also been shown to increase H3K9me3 levels in the hippocampus [130]. This
repressive mark appears to accumulate selectively at repetitive elements, specifi-
cally retrotransposons (for review see Lapp & Hunter, 2016) in the genome [131,
132]. Interestingly, Alu and LINE1 retrotransposons appear upregulated in PTSD
veterans compared to combat deployed controls [133]. Socially defeated animals
also show increased basal corticosterone in circulation, reduced time spent in open
arms of the EPM and in the light component of the light/dark box, as well as reduced
A.A. Bartlett et al.
157
hippocampal H3 acetylation and increased HDAC5 expression [134]. These defi-
cits, however, were rescued by a moderate, involuntary exercise regiment, a mild
stressor [134]. Voluntary exercise, a mild and controllable stressor, alone has been
shown to have anxiolytic effects in addition to reducing hippocampal expression of
the histone H2 variant H2A.z and increasing expression of mitochondrial- related
genes TFAM and NDUFA6 in the same region [135]. These recent findings hark
back to the importance of allostasis and suggest an epigenetic underpinning of anx-
ious behaviors. Further, it has been suggested that stress opens up “windows of
epigenetic plasticity” that are unique to the stressor and elicit dynamic effects based
on previous stress history [136, 137]. The recent work of the McEwen laboratory
has provided strong evidence for this nuanced view of the epigenetic effects of
stress. While chronic restraint stress resulted in reduced time spend in the light
component of the light/dark box, only a novel acute stressor led to persistent reduc-
tion in time spend in the light compartment. These differences corresponded to
changes in hippocampal long-term potentiation and NMDA receptor expression
[136, 137]. Acute restraint stress exposure has also been shown to convert DNA
methylation through the addition of a hydroxyl group of NR3C1 promoter in the
hippocampus [138]. More recently, hyper-hydroxymethylation has been observed
in regions associated with neuronal plasticity following acute restraint stress in the
hippocampus [139].
8.9 Prospects for an Epigenetic Pharmacology of Anxiety
Epigenetic interventions have proven effective in animal models of anxiety and
stress, and some psychiatric drugs, such as the mood stabilizer valproate, have
known epigenetic effects (valproate is an HDAC inhibitor). Thus, it would appear
that the prospects for epigenetic therapies for anxiety disorders are fairly high.
Most pharmacologic studies of drugs with epigenetic activities have focused on
histone acetylation, with the HDACs being the major targets. In fear extinction
models, which have substantial relevance to human AD, a variety of HDAC inhibi-
tors have been shown to be effective in enhancing extinction [140]. Similarly,
HDAC inhibition reversed the group differences in maternal behavior and adult
stress reactivity observed by Weaver in his landmark paper on epigenetic program-
ming of maternal behavior [33]. Similarly, the same phenotype and associated anx-
ious behavior could be reversed with central infusion of the methyl donor
S-adenosyl-methionine (SAMe) in adult animals [34, 35]. A number of studies have
found SAMe to be more effective than placebo in the treatment of depression,
though other well-designed trials have had negative results [141, 142]. A recent
Cochrane collaboration review concluded that there was not strong evidence for the
efficacy of SAMe in depression but that further research was warranted [143].
Studies of the efficacy of SAMe in the treatment of anxiety symptoms, however, are
very limited to date. DNA methyltransferase inhibitors such as zebularine,
N-phthaloyl-l-tryptophan and 5-aza-deoxycytidine have been shown to interfere
with fear memory formation in preclinical models [66, 118]. To date, little clinical
8 Anxiety and Epigenetics
158
work has been done with this class of drugs, likely due to concerns about side
effects, which are significant for some of these agents.
The study of epigenetic drug targets for anxiety remains in its infancy, and many
questions remain to be adequately researched. One such question is whether these
agents actually offer superior outcomes to existing treatments. Another is whether
they might be used in combination with both other drugs and behavioral interven-
tions to additive or even synergistic effect. Nonetheless, molecular epigenetics
offers a novel class of potential drug targets for disorders like AD which have his-
torically had relatively few molecular mechanisms with which to work.
Conclusions
Epigenetic mechanisms play a clear mechanistic role in animal models of anxi-
ety, and human epigenetic studies suggest that these observations are generaliz-
able to clinical populations. Indeed, some effort has already been made to
translate the preclinical findings in the field into the clinic. Nonetheless, signifi-
cant questions, particularly those relating to the time course and nature of epi-
genetic changes in humans, remain to be answered. Beyond the borders of what
might now be regarded as “classical” epigenetics, novel molecular mechanisms
of epigenomic, genomic, and epitranscriptomic plasticity are being revealed in
the brain in behavioral contexts relevant to anxiety disorders. Transposons,
which are mobile elements of the genome, have been shown to be regulated by
stress exposure in both humans and animal models [131, 144, 145]. The mito-
chondria, which contains its own genome, shows transcriptional regulation in
response to stress, and its function in the nucleus accumbens has been linked to
anxiety phenotypes and social subordination in mice [146–148]. Even more
intriguingly, covalent modification of RNA in the prefrontal cortex, the methyla-
tion of adenosine, has been shown to associate with the development of fear
memory in mice [149]. This epitranscriptomic effect points to yet another layer
of molecular complexity that will need to be incorporated into our models of
anxiety, both normal and pathologic in model systems and in the clinic.
While neuroepigenetics is a relatively young science, it is already clear that it
has relevance to our understanding of AD. Indeed, it has begun to produce usable
translational findings for the treatment of disorders, like depression, which are
highly comorbid with numerous anxiety disorders. There is ample reason to
believe that neuroepigenetic mechanisms will continue to be a fruitful area of
research into the biology of anxiety and AD.
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