Involvement of Epigenetic Modifications of GABAergic Interneurons in Basolateral Amygdala in Anxiety-like Phenotype of Prenatally Stressed Mice
Advance Access Publication: February
Involvement of Epigenetic Modifications of GABAergic Interneurons in Basolateral Amygdala in Anxiety-like Phenotype of Prenatally Stressed Mice
Chunting Zhu 0 1
Min Liang 0 1
Yingchun Li 0 1
Xuejiao Feng 0 1
Juan Hong 0 1
Rong Zhou 0 1
0 Province , China 211166
1 Department of Physiology, Nanjing Medical University , Jiangsu , China
Background: Prenatal stress is considered a risk factor for anxiety disorder. Downregulation in the expression of GABAergic gene, that is, glutamic acid decarboxylase 67, associated with DNA methyltransferase overexpression in GABAergic neurons has been regarded as a characteristic component of anxiety disorder. Prenatal stress has an adverse effect on the development of the basolateral amygdala, which is a key region in anxiety regulation. The aim of this study is to analyze the possibility of epigenetic alterations of GABAergic neurons in the basolateral amygdala participating in prenatal stress-induced anxiety. Methods: Behavioral tests were used to explore the prenatal stress-induced anxiety behaviors of female adult mice. Real-time RT-PCR, western blot, chromatin immunoprecipitation, and electrophysiological analysis were employed to detect epigenetic changes of GABAergic system in the basolateral amygdala. Results: Prenatal stress mice developed an anxiety-like phenotype accompanied by a significant increase of DNA methyltransferase 1 and a reduced expression of glutamic acid decarboxylase 67 in the basolateral amygdala. Prenatal stress mice also showed the increased binding of DNA methyltransferase 1 and methyl CpG binding protein 2 to glutamic acid decarboxylase 67 promoter region. The decrease of glutamic acid decarboxylase 67 transcript was paralleled by an enrichment of 5-methylcytosine in glutamic acid decarboxylase 67 promoter regions. Electrophysiological study revealed the increase of postsynaptic neuronal excitability in the cortical-basolateral amygdala synaptic transmission of prenatal stress mice. 5-Aza-deoxycytidine treatment restored the increased synaptic transmission and anxiety-like behaviors in prenatal stress mice via improving GABAergic system. Conclusion: The above results suggest that DNA epigenetic modifications of GABAergic interneurons in the basolateral amygdala participate in the etiology of anxiety-like phenotype in prenatal stress mice. Introduction effect of early-life stress on brain development. For example, epiBrain development is an intricate and subtle process and greatly demiologic studies have indicated that children exposed to early sensitive to stress and environmental factors. Clinical and pre- adverse experiences are at increased risk for the development clinical studies have indicated that early-life stress including of depression and anxiety disorders in the adulthood (Heim prenatal stress (PRS) has an adverse effect on the neurodevel- and Nemeroff, 2001; Cartwright-Hatton, 2006). Consistently, opment leading to the permanent abnormality of brain struc- animal studies have indicated that prenatally stressed rats or ture and function. Anxiety disorder in childhood or adulthood mice exhibit excessive anxiety-like behaviors in the elevated is regarded as the common long-term outcome of the disrupted plus maze (EPM) and the open field test (OFT) (Vallee et al., 1997;
anxiety; DNMT1; epigenetic; GABAergic dysinhibition; GAD67; prenatal stress
The present study provides new insight into investigating the mechanisms underlying the influence of prenatal stress (PRS)
on adult behaviors. Since PRS mice shows a similar epigenetic signature to patients with anxiety disorder, PRS mice can be a
valid animal model to study the epigenetic mechanisms underlying anxiety disorder and validate the novel antianxietic drugs.
Furthermore, our study reveals the validity of DNA methyltransferase 1 (DNMT1) inhibitor in improving a variety of PRS-induced
anxiety alterations. DNMT1 may represent a potential new molecular target to treat patients with anxiety disorder. Thus far,
epigenetic drugs have been used in cancer treatment, because they often selectively reactivate tumor suppressor genes that are
silenced by CpG island promoter methylation.
Mairesse et al., 2007;Zuena et al., 2008;Papale et al., 2017) and the NIH Guide for the Care and Use of Laboratory Animals. All
an increased reactivity of hypothalamo-pituitary-adrenal (HPA) efforts were made to minimize the number of animals and their
axis to stress indicative of a typical anxious phenotype that is suffering.
observed in anxiety patients (Darnaudery and Maccari, 2008;
Weinstock, 2008). Animal Model Preparation
Abundant data have demonstrated that “persistency” is the
most obvious characteristic of the effects of early-life stress. Pregnant C57BL/6J mice (Oriental Bio Service Inc) were
individuAlthough the mechanisms underlying the relationship between ally housed and food and water ad libitum. Control dams were
adverse developmental experiences and life-long phenotypic left undisturbed throughout gestation with a 12-h-light/-dark
consequences are unclear, the epigenetic role of early-life cycle (lights on at 7:00 am and off at 7:00 pm), whereas stressed
stress in the pathogenesis of anxiety disorder was suggested dams were subjected to the restraint stress as described
prein epidemiological studies (Murgatroyd et al., 2009M; eaney and viously (Zheng et al., 2016) with slight modification. From the
Ferguson-Smith, 2010; Papale et al., 2017). An early-life stressor tenth day of pregnancy until delivery, the pregnant dams were
has been reported to increase the expression of DNA methyl- restrained in a transparent tube (12 cm× 3 cm) for 45 min 3
transferase (DNMT1 and 3a) in the postnatal brain (Matrisciano times/d and exposed to 24-h constant light. The day of birth was
et al., 2013;Dong et al., 2015). DNMT1 and DNMT3a are mainte- referred to as postnatal day 0 (PND 0). Weaning occurred at PND
nance enzymes that selectively localize in GABAergic neurons, 21 to 22, after which the offspring of control and stressed dams
and the overexpression of these enzymes has been reported (control and PRS mice) were group-housed by litter and sex. To
in GABAergic neurons of cortical or striatal brain areas in psy- minimize the effect of parent-to-offspring interaction per litter,
chotic patients (Veldic et al., 2005V;eldic et al., 2007;Zhou et al., one female offspring was randomly selected from each litter as
2013). The promoter of glutamic acid decarboxylase (GAD) 67, object of study. Male offspring were not taken into experiments,
which is one of GABAergic neuronal markers, has been proved because the anxiogenic effects of PRS were mainly observed in
to be embedded in large CpG islands and express methylation females but not males (Papale et al., 2017). Then, the following
(Grayson et al., 2005;Costa et al., 2007)
. Decreased experiments were performed at PNDs 60 to 70.
GAD67 expression and increased DNMTs expression, as well an
increase in the methylation of GAD67 promoter, have recently Behavior Analysis
been reported in the brain of adult mice prenatally exposed to
stress (Matrisciano et al., 2012, 2013). To avoid the influence of the fluctuation of gonadal hormones
The GABAergic inhibitory circuit in the basolateral amyg- in estrous cycle on anxious behaviors
(Koonce et al., 2012)
dala (BLA) has been identified as a key regulator of anxiety- behavioral experiments were performed at diestrus of female
like behaviors in both preclinical and clinical settings (Sanders mice. According to the types of vaginal epithelium cells
(leukoand Shekhar, 1995; Allison and Pratt, 2003B;ueno et al., 2005; cyte cells, nucleated cells, and cornified cells)
Ackermann et al., 2008
). The deficit in GABAergic system in the trus, and estrus were determined. Mice were used to examine
BLA is implicated in the pathophysiology of neurodevelopmen- anxiety-like behaviors on 3 mornings following OFT (on the first
tal psychiatric disorders (Quirk and Gehlert, 200B3e;nes, 2010; morning)→EPM (on the second morning)→light/dark box test
Felix-Ortiz et al., 2013). Several studies have identified effects of (DLT on the third morning) sequence. The room was dimly lit
PRS on the developing amygdala (Kraszpulski et al., 2006C;haril during the tests. The detailed methods of the behavioral tests
et al., 2010) and adult amygdala function (Weinstock, 2008; have been described previously
(Chiba et al., 2012)
Sadler et al., 2011). behaviors were videotaped and quantified via Ethovision 3.0
Considering that PRS has adverse effects on DNA methylation software (Noldus Information Technology Inc.).
and the development of amygdala, we investigated whether PRS
results in the increase of anxiety-related behaviors through an OFT
epigenetic GABAergic dysfunction in the BLA. The aim of the pre- The novel environment was a 35 cm × 35 cm × 25 cm white
sent study is to increase the understanding of epigenetic mecha- Plexiglas arena. Mice were placed in a corner and were allowed
nisms underlying anxiety disorder by analyzing the interactions to freely explore the field for 10 min. Both total traveled distance
between genes and the environment in an animal model. and time spent in the center area were recorded.
Materials and Methods
The present studies were approved by the Animal Care and Use Committee of Nanjing Medical University. The protocols used here were in accordance with the guidelines published in
The elevated plus maze consisted of 2 open and 2 closed arms
with each arm projecting 50 cm from the center (a 10× 10 cm
area). The whole apparatus was placed 50 cm above the floor.
Control and PRS mice were placed in the center of the EPM,
facing a closed arm, and were allowed to freely explore the maze
for 10 min. The number of 4-paw entries and the time spent in
the 2 open arms were blindly analyzed.
For protein quantification, we conducted measurements as
described in detail elsewhere (Matrisciano et al., 2012). The
DLT BLA was dissected on ice and the protein was extracted using
The DLT test was performed in a rectangular box divided in a total protein extraction kit (KGP250KGP2100; KeyGEN Biotech).
2 compartments (light and dark). A removable dark Plexiglas Anti-DNMT1 monoclonal antibodies (NB100-264; Imagenex) and
partition was used to divide the box into light and dark sides. anti-GAD67 polyclonal antibody (MAB5406; Millipore) were used
Each animal was placed into the light side of the box, facing to detect DNMT1 and GAD67 protein. Anti-β-actin monoclonal
away from the dark side and allowed to explore both chambers antibody (04-1116; Millipore) was used an internal antibody. The
of the apparatus for 10 min. The time spent in the light side IMAGEQUANT analysis software was used to perform the
densiand the number of entries into the dark compartment were tometric analysis of interest bands. The values were presented
scored. as an optical density ratio with respect to β-actin.
Chromatin Immunoprecipitation Assays
Two batches of mice were killed, one between 7:30 and 8:00 am Chromatin immunoprecipitation (ChIP) assays were used to
and the other between 5:30 and 6:00 pm. Plasma was separated detect DNMT1 or MeCP2 binding to CpG-rich regions of GAD67
and stored at -20°C for hormones assay. The concentrations of promoter. The Chip procedure was carried out using a ChIP kit
plasma corticosterone and adrenocorticotropin (ACTH) were (17–295; Upstate). Briefly, approximate 10 mg of the BLA was
assayed by a 125I double-antibody radioimmunoassay kit (07- used for this procedure. Tissue was incubated at 37°C for 10 min
189105; MP Biomedicals) according to the instructions of the with 500 mL of PBS containing 1% formaldehyde and a cocktail
manufacturer. The ACTH assay has a sensitivity of 2 pg/mL and of protease inhibitors (P8340; Sigma). Tissue was homogenized
intra-assay and interassay CVs of <8%. The corticosterone assay in 300 mL of SDS lysis buffer (supplied by ChIP kit, Upstate), and
has a sensitivity of 1 ng/mL and intra-assay and interassay CVs the lysate was sonicated for 15 min on ice. Immunoprecipitation
of <10%. was performed overnight at 4 °C by the addition of 10μ g of ChIP
grade DNMT1 (ab87656; Abcam) or MeCP2 (ab2828; Upstate) to
Real-time RT-PCR the sonicated solution. An aliquot of the sonicated lysate
without antibody was used as input to quantify the total amount
Mice were killed (2 h after lights on) and whole brains were of DNA in sample extracts. Protein-free DNA was extracted and
extracted and immediately stored at -80°C until assayed. Brains used for detection and quantification of CpG-rich regions of
sections (50 μm thick) were cut in the coronal plane using a GAD67 promoter by quantitative PCR. The primers of CpG-rich
cryostat. The BLA subfield was dissected from the frozen slices GAD67 promoter were decided by the reports of Matrisciano
on dry ice. Total RNA was extracted using TRIzol (15596–026; et al., (2013) and shown inTable 1. The level of
immunoprecipiInvitrogen) following the manufacturer’s instructions. Possible tated GAD67 promoter by the DNMT1 or MeCP2 antibody was
contamination with genenomic DNA was removed by an on- expressed as a percentage of the input DNA using the following
column DNase I (15200, Qiagen) treatment. mRNA was reverse equation: % (DNA-IP/total input=) 2 [(Ct(10%input) – 3.32) – Ct
transcribed using the high-capacity cDNA Reverse Transcription (DNA-IP)] × 100%.
kit (4368814; Applied Biosystems) following the manufactu-r
er’s instructions. The primer sequences of DNMT1, DNMT3a, Methylated/Hydroxymethylated DNA
DNMT3b, methyl CpG binding protein 2 (MeCP2), ten-eleven Immunoprecipitation
translocation 1 (TET1), GAD67, and GAPDH are shown iTnable 1.
These primer sequences were decided upon according to the pre- Methylated (5MC) and hydroxymethylated DNA (5HMC) on
vious reports (
La Salle et al., 2004
M; atrisciano et al., 2013D;ong GAD67 promoter were assessed using MeDIP (C02010011;
et al., 2015). RT-PCR was performed using a LightCycler FastStart Diagenode) and hMeDIP kits (C02010034; Diagenode) followed
DNA Master SYBR Green I kit (3515869001; Roche) and an ABI by quantitative PCR. The procedures for sample treatment and
Prism 7300 Sequence Detection System (Applied Biosystems). immunoprecipitation are described in the kit instruction
manuTo improve the accuracy of the real-time PCR for quantification, als. The following quantitative PCR, the primers of GAD67
proamplifications were performed intriplicate for each RNA sample. moter, and the calculation of the level of immunoprecipitated
The relative expression of genes was determined using the 2-ΔΔCt GAD67 promoter by the 5MC or 5HMC antibody were the same
method with normalization to GAPDH expression. as those in ChIP assays.
Data were retrieved and processed with the Origin 6.1 software
(Micro-Cal Software Inc.). The group data were expressed as the
means ±SEM. Two-tailed Student’st test was used for
comparisons between 2 groups. ANOVAs followed by Fisher’s protected
Field Potential Recording least significant difference (PLSD) posthoc test was employed
For recording, the BLA slices were transferred to a chamber con- if more than 2 groups were compared. Statistical analysis
tinuously perfused with oxygenated ACSF (2 mL/min) maintained was performed using Stata 7 software (Stata Corp). P < .05 was
at 30°C. Biphasic square wave pulses were applied at EC through considered statistically significant. For statistical purposes,
a stainless-steel stimulation electrode (Figure 6A). Stimulation- only one slice was studied per mouse in electrophysiological
evoked population spikes (PS) were recorded from the BLA by a analysis.
glass micropipettes filled with 2 M NaCl (4–5 M)Ω connected to an
Axoclamp2B amplifier (Axon Instruments). PS response was
sampled using pCLAMP software (Axon Instruments). The PS ampli- RESULTS
tude was defined as the mean amplitude of the peak negativity,
measured from the peak of the early and the late negativity. Paired- PRS Mice Express the Increase of Anxiety-like
pulse inhibition (PPI) of the PS was evoked by double-pulse stimu- Behaviors in Behavioral Experiments
lation to EC at 50% of maximal stimulus strength and expressed
as the ratio of the second PS amplitude to the first one. The int-er
val between double pulses (20, 50, 75, 100 ms) was adopted to fall OFT
within the time course of the intracellularly recorded IPSP attrib- PRS mice showed a decrease in time spent in the central area
uted to activation of GABAergic interneurons (Rainnie et al., 1991). of the open field in comparison with age-matched controls
(t(26) = 2.41, P < .05; Figure 2A). To exclude the influence of
locomoDrug Administration tion abnormality in this test, we also measured the locomotor
activity of those mice. No significant difference in total traveled
Cannulae were implanted with bilateral 27-gauge stainless steel distance between control and PRS groups was observed in the
cannulas into the bilateral BLA (Figure 1) according to the mouse present study ( t(26) = 0.27, P > 0.05; Figure 2B).
The percentage of time spent in the open arms was decreased
in PRS offspring as compared with controls ((t26) = 3.08, P < .01;
Figure 2C). However, the number of 4-paw entries in the test area
was not affected by PRS (t(26) = 0.48, P > 0.05; Figure 2D).
from control and PRS mice at circadian nadir (8:00 am) and
peak (6:00 pm). As shown in Figure 3A–B, PRS mice exhibited
a significant increase in both basal and peak corticosterone
release compared with controls (basal:(2t0) = 2.51, P < .05; peak:
t(20) = 2.74, P < .05). Similarly, PRS mice had increased basal plasma
ACTH and exhibited significantly increased peak ACTH (basal:
t(20) = 2.32, P < .05; peak: t(20) = 2.21, P < .05).
PRS mice spent less time in the light-box of the DLT than con- DNMT1 Excess Is Accompanied by the Decreased
trols ((t26) = 2.26, P < .05; Figure 2E). No statistically significant dif- GAD67 Expression in the BLA of PRS Mice
ferences were found in terms of the total number of transitions
between the 2 groups ( t(26) = 0.146, P > 0.05; Figure 2F).
Earlier reports suggest that an increase in DNMT levels is asso
ciated with a downregulation of GAD67 in postmortem brain
PRS Results in the Potentiation of Circadian HPA- tissue from patients with schizophrenia or bipolar disorders
Axis Activity (Guidotti et al., 2011) and in brain of early postnatal stressed
rats (Zhang et al., 2010). The results of real-time RT-PCR showed
To test whether PRS affects HPA-axis regulation leading to the that in several detected enzymes associated with DNA
methchange of anxiety-related behaviors, plasma was collected ylation modification, only the expression of DNMT1 mRNA
was significantly higher in the BLA of PRS mice compared with in PRS mice was significantly greater than that for the control
control mice (t(14) = 3.16, P < .01; Figure 4A). The result of western group (t(18) = 4.21, P < .01). Based on the previous studies (Wang
blot indicated that associated with an increased level of DNMT1 et al., 2001,2002), the appearance of PSs suggests the possibility
(t(14) = 4.29, P < .01), PRS mice showed a marked decrease in GAD67 that the inhibitory mechanisms that normally restrict repetitive
protein level in the BLA ((t14) = 2.41, P < .05; Figure 4B) compared firing in the BLA are impaired in PRS mice. PPI with inter-pulse
with control mice. intervals (IPIs) ranging from 20 to 100 ms were then introduced
to determine the inhibitory activity of GABAergic interneuron
DNMT1-Induced Hypermethylation of GAD67 in the BLA
(Isoardi et al., 2004)
. As shown in Figure 6C, 2-way
Promoter Results in the Downregulation of GAD67 in repeated ANOVA indicated statistically significant effects with
the BLA of PRS Mice PRS (F(1,20) = 58.024; P < .001), IPIs (F(1,20) = 5.373; P < .05), and PRS by
IPI interaction (F(1,20) = 7.916; P < .05). PPI was mainly observed
To test whether the overexpression of DNMT1 in PRS mice co-r with IPIs at 20 to 75 ms in the control mice, whereas
pairedrelates with an increased binding of DNMT1 to specific GAD67 pulse facilitation (PPF) instead of PPI was evoked by 20 to 75 ms
CpG-rich promoter sequences, we measured the binding of IPIs in PRS mice (P < .01). The PPI-reversed to PPF in PRS mice
DNMT1 to GAD67 promoter by ChIP assay. The results showed proves the fact that the decrease of GABAergic inhibitory
mechthat the binding of DNMT1 to GAD67 promoter region was anism results in the increase of the BLA neuronal excitability.
increased in PRS mice (t(10) = 3.24, P < .01; Figure 5A). Moreover, the Two-way ANOVA displayed main effects of PRS and 5-aza-CdR
binding of MeCP2 to GAD67 was also significantly higher in PRS treatment and their interaction on the number of PSs (PRS:
mice (t(10) = 2.68, P < .05; Figure 5B) in the absence of the expres- F(1,42) = 28.62; P < .001; 5-aza-CdR: F(1,42) = 34.35; P < .001;
PRS×5-azasion change of MeCP2 mRNA. CdR: F(1,42) = 38.01; P < .001; Figure 6D) and PS2/PS1 (PRS: F(1,42) = 5.54;
To investigate whether the decreased expression of GAD67 P < .05; 5-aza-CdR: F(1,42) = 4.72; P < .05; PRS×5-aza-CdR: F(1,42) = 4.384;
in PRS mice are the consequence of epigenetic modification on P < .05; Figure 6E). The followed PLSD posthoc test further
indithe corresponding DNA regulatory regions, we measured the cated that treatment with 5-aza-CdR abolished the repetitive
enrichment of the 2 most important epigenetic marks: 5MC response (P < .01) and recovered PPI (P < .01) in the slices from
and 5HMC at the GAD67 promoter. As shown in Figure 5C, PRS offspring, suggesting the involvement of DNMT1 in the BLA
5MC was enriched at GAD67 promoter in PRS mice compared GABAergic deficiency.
with control mice ((t10) = 2.66, P < .05). The 5HMC level in PRS
mice was higher than control mice, but did not reach the
significant level ((t10) = 1.04, P > 0.05; Figure 5D). These findings The Intra-BLA Injection of 5-aza-CdR Corrects
suggest that PRS leads to CpG hypermethylation on GAD67 Anxiety-like Behavior in PRS Mice
promoters. The enrichment of 5MC at GAD67 promoter was To test whether the behavioral alterations of PRS mice were
negatively correlated with the level of corresponding GAD67 mediated by epigenetic mechanisms including an increase in
transcript (2r= 0.352, P = .03; Figure 5E), suggesting an epigenetic DNMT1, an increase of GABAergic promoter methylation, and
mechanism by which promoter methylation may be respon- a downregulation of the GABAergic gene expression, the
behavsible for the downregulation of GAD67 in PRS mice ((1t0) = 3.83, ior of PRS mice was evaluated following the repeated
microinP < .01; Figure 5F), and this possibility was therefore tested via jection of 5-aza-CdR into the BLA. Two-way ANOVA revealed a
detecting the effect of repeated intra-BLA injection of 5-aza- main effect of PRS (center time: F(1,60) = 4.83, P < .05; time in the
CdR, DNMT1 inhibitor on 5MC at GAD67 promoter, and GAD67 open arm: F (1,60) = 5.15, P < .05; time in light: F(1,60) = 4.32, P < .05) and
transcript. Two-way ANOVA displayed main effects of PRS and 5-aza-CdR treatment (center time: (F1,60) = 5.36, P < .05; time in
5-aza-CdR treatment and their interaction on 5MC at GAD67 the open arm: F(1,60) = 5.56, P < .05; time in light: F(1,60) = 5.73, P < .05)
promoter (PRS: F(1,24) = 6.28; P < .05; 5-aza-CdR: F(1,24) = 4.89; P < .05; and a PRS×5-aza-CdR treatment interaction effect (center time:
PRS×5-aza-CdR: F(1,24) = 4.71; P < .05; Figure 5G) and GAD67 tran- F(1,60) = 4.95, P < .05; time in the open arm: F (1,60) = 8.64, P < .05; time
script (PRS: F(1,24) = 5.77; P < .05; 5-aza-CdR: F(1,24) = 5.01; P < .05; in light: F(1,60) = 3.02, P < .05). The results of the followed PLSD
postPRS×5-aza-CdR: F(1,24) = 8.53; P < .01; Figure 5H). The treatment hoc test further showed that 5-aza-CdR rectified the changed
with 5-aza-CdR abolished both the alterations of 5MC level at center time in the OFT (P < .05;Figure 7A), time spent in the open
GAD67 promoter (P < .05) and GAD67 transcript (P < .01) in PRS arm of the EPM (P < .05; Figure 7B), and time in the light of the DLT
mice. (P < .05; Figure 7C), but had no effect in control mice (P > 0.05).
DNMT1-Mediated GABAergic Dysfunction
Participates in the Enhancement of Cortical-BLA
Transmission in PRS Mice
This study represents the first demonstration that PRS facili
tates anxiety-like behaviors and attenuates GABAergic
inhibiGABAergic system in the BLA plays a key role in inhibiting the tion in the BLA of female mice offspring, which is at least partly
excitation of the pyramidal cells that receive cortical glutam-ater via DNMT1-related epigenetic reprogramming of GABAergic
sysgic inputs (Stell et al., 2003M;aguire et al., 2005). Therefore, to tem. In addition, the present data also suggest the long-term
evaluate the possibility that epigenetic modification of DNMT1 neurobehavioral effects of PRS are reversible in the adult period.
further changes GABAergic function in the BLA of PRS mice, It has been widely proved that early life stress causes
longcortical-BLA glutamate synaptic transmission was detected lasting changes in neuroplasticity that result in an increased
(Figure 6A). As shown in Figure 6B, in slices from control off- vulnerability to stress-related disorders in later life
spring, a moderate single pulse evoked a PS generated by the et al., 2007;Darnaudery and Maccari, 2008;Lupien et al., 2009)
synchronous firing of basolateral projecting neurons. By con- The present data that PRS resulted in anxiety-like behaviors
trast, in slices from PRS mice, the same manner of stimulation and associated endocrinological alterations of adult female
evoked the appearance of several additional responses follow- mice give further support to the above notion. Female mice
ing the main PS in the BLA neurons. The average number of PSs exposed to stress in utero represent a new behavioral model of
an anxiety-like phenotype, because it recapitulates the potential
link between early-life adversity and the pathogenesis of
The early development period is crucial for establishing
and maintaining epigenetic marks (Reik et al., 2001;Reik, 2007).
Epigenetic mechanisms are consequently regarded as the most
plausible targets through which early life stress could exert their
long-lasting effects. Indeed, accumulating evidence has proved
the DNA epigenetic alterations induced by prenatal restrained
stress contribute to the complex phenotypes of
neuropsychiatric disorders (Matrisciano et al., 201Z2;heng et al., 2016;
Papale et al., 2017). DNMTs are important components of the
DNA-methylation that dynamically regulates the expression of
key molecules involved in brain function. Here, we focused on
DNMTs, because these enzymes have been proved to participate
in the physiopathology of several neurodevelopmental diso-r
ders including anxiety disorder (Ruzicka et al., 200V7;eldic et al.,
2007; Guidotti et al., 2011). Our results showed that in these
detected enzymes, only the expression of DNMT1 was found to
be elevated in the BLA of adult female PRS offspring. To establish
in detail whether the altered expression of DNMT1 is expected
to result in enrichment of CpG methylation at GAD67
promoters, we then measured levels of 5mC, a CpG methylation marker.
As expected, there was significant methylation (high levels of
5mC) found on GAD67 promoters in female PRS mice, and the
increased promoter methylation of GAD67 was inversely co-r
related with the corresponding transcripts. These data suggest
PRS results in DNMT1-regulated hypermethylation of GAD67
promotor, thereby inhibiting GAD67 transcription in the BLA.
However, we cannot exclude the possibility that histone
modifications, another epigenetic regulation of gene transcription,
could contribute to the epigenetic modifications detected in
female PRS mice.
The GABAergic system in the BLA participates in the
regulation of emotional behaviors via inhibiting the excitation of the
pyramidal cells that receive cortical glutamatergic inputs (Stell
et al., 2003;Maguire et al., 2005). Research in our laboratory and
others have established the appearance of multiple PSs in co-r
tical-BLA transmission due to the decreased GABAergic
inhibitory effect on the pyramidal neurons (Rodriguez Manzanares
et al., 2005). In the present study, the electrophysiological
finding that multiple PSs instead of single PS were measured in the
BLA slices of female PRS mice strongly suggests that PRS results
in the BLA GABAergic deficits. This inference also is supported
by the fact that PPI reversed into PPF in the slices of female PRS
mice. Guidotti et al. (2005) have found that there is an obvious
decrease of GAD67 and other markers of GABAergic
interneurons but not neuronal loss in the postmortem brains of patients
with neurodevelopmental psychiatric disorders. The present
study shows that the overexpression of DNMT1 is responsible
for the decrease of GAD67. Hence,it is plausible that
DNMT1of DNMT1 in the BLA, it is possible to analyze the role of DNMT1 female PRS mice. Our study indicates PRS leads to the increase of
in long-lasting anxiety using pharmacological methods. In fact, anxiety level in female offspring through epigenetic GABAergic
we have found that the local blockade of DNMT1 with 5-aza-CdR dysfunction. However, another report byMatrisciano et al.
in the BLA significantly ameliorated anxiety-like symptoms in (2013) has pointed out the involvement of PRS-induced
epigenPRS mice. It is noteworthy that the same dosage of 5-aza-CdR etic modifications of GABAergic interneurons in schizophrenia
as that used in PRS mice fails to change anxiety-related behav- in male mice. These data suggest that PRS-induced epigenetic
iors of control mice, suggesting a specificity of action on the epi- DNA alternations have a sexual dimorphic effect on behavioral
genetic mechanisms that underlie the behavioral pathology in outcome. A review by
Charil et al. (2010
) has pointed out that the
sexual dimorphic effect of PRS is related with the sex-difference
in brain development. It is therefore inferred that the sex-diff-er
ence in brain development may be a key factor responsible for
sex dimorphisms in neuropsychiatric disorders associated with
PRS-induced epigenetic DNA alternations and this possibility
requires further investigation.
It has been found that there is a persistent overexpression
of DNMT in the brain of PRS mice ranging from birth to
adulthood (Matrisciano et al., 2013). We can infer that in female PRS
mice, the increase of DNMT1 is probably the result of changes
occurring during embryonic life. Further, stress exposure du-r
ing pregnancy results in the decrease of maternal cares on
pups (Pardon et al., 2000). Previous studies suggest that the
variations of maternal care have a stable effect on
anxietymediated behaviors through epigenetic mechanisms (Weaver
et al., 2006). Therefore, the change of maternal care could be
another factor involved in DNMT1 upregulation in female
In conclusion, these preclinical studies in mice support the
concept that the PRS model has construct face validity and
pharmacological utility as an experimental epigenetic model of
anxiety disorders. We propose that the upregulation of DNMT1
leads to the hypermethylation and increased binding of DNMT1
to GAD67 promoters in PRS model. Furthermore, we propose
that drugs that induce promoter hypomethylation and/or
DNMT1 downregulation might be useful in correcting anxious
behaviors. This means that DNMT1 may represent possible new
molecular targets to treat with the long-term neurobehavioral
effects of developmental stress exposure.
This work was supported by the National Natural Science Foundation of China (81471385) and the Natural Science Foundation of Jiangsu Province of China (BK20151552).
Statement of Interest
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