Epigenetic modifications of GABAergic interneurons are associated with the
schizophrenia-like phenotype induced by prenatal stress in mice
Francesco Matriscianoa,b,*, Patricia Tuetinga, Ishani Dalala, Bashkim Kadriua, Dennis R. Graysona,
John M. Davisa, Ferdinando Nicolettib,c, Alessandro Guidottia
aThe Psychiatric Institute, Department of Psychiatry, College of Medicine, The University of Illinois at Chicago, Chicago, IL 60612, USA
bDepartment of Physiology and Pharmacology, University of Rome “Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy
cI.N.M. Neuromed, Pozzilli, Italy
a r t i c l e i n f o
Received 17 January 2012
Received in revised form
26 March 2012
Accepted 13 April 2012
a b s t r a c t
Human studies suggest that a variety of prenatal stressors are related to high risk for cognitive and
behavioral abnormalities associated with psychiatric illness (Markham and Koenig, 2011). Recently,
a downregulation in the expression of GABAergic genes (i.e., glutamic acid decarboxylase 67 and reelin)
associated with DNA methyltransferase (DNMT) overexpression in GABAergic neurons has been regarded
as a characteristic phenotypic component of the neuropathology of psychotic disorders (Guidotti
et al., 2011).
Here, we characterized mice exposed to prenatal restraint stress (PRS) in order to study neurochemical
and behavioral abnormalities related to development of schizophrenia in the adult. Offspring born from
non-stressed mothers (control mice) showed high levels of DNMT1 and 3a mRNA expression in the
frontal cortex at birth, but these levels progressively decreased at post-natal days (PND) 7, 14, and 60.
Offspring born from stressed mothers (PRS mice) showed increased levels of DNMTs compared to
controls at all time-points studied including at birth and at PND 60. Using GAD67-GFP transgenic mice,
we established that, in both control and PRS mice, high levels of DNMT1 and 3a were preferentially
expressed in GABAergic neurons of frontal cortex and hippocampus. Importantly, the overexpression of
DNMT in GABAergic neurons was associated with a decrease in reelin and GAD67 expression in PRS mice
in early and adult life. PRS mice also showed an increased binding of DNMT1 and MeCP2, and an increase
in 5-methylcytosine and 5-hydroxymethylcytosine in specific CpG-rich regions of the reelin and GAD67
promoters. Thus, the epigenetic changes in PRS mice are similar to changes observed in the post-mortem
brains of psychiatric patients. Behaviorally, adult PRS mice showed hyperactivity and deficits in social
interaction, prepulse inhibition, and fear conditioning that were corrected by administration of valproic
acid (a histone deacetylase inhibitor) or clozapine (an atypical antipsychotic with DNA-demethylation
activity). Taken together, these data show that prenatal stress in mice induces abnormalities in the
DNA methylation network and in behaviors indicative of a schizophrenia-like phenotype. Thus, PRS mice
may be a valid model for the investigation of new drugs for schizophrenia treatment targeting DNA
This article is part of the Special Issue entitled ‘Neurodevelopmental Disorders’.
Published by Elsevier Ltd.
Brain development comprises a time- and region-specific
complex sequence of neuronal events, involving neurogenesis
and neuronal differentiation that result in the generation of the
normal structure and function of the adult brain. It is now accepted
that stress and environmental factors can lead in adulthood to
neuropsychiatric disorders including schizophrenia (SZ) and
bipolar (BP) disorders by disrupting the sequence of these neuro-
developmental events (Howes et al., 2004; Weinberg and Lipska,
1995; Lewis and Levitt, 2002; Barker, 2003; Rapaport et al.,
2005). For example, the exposure of pregnant women to psycho-
logical stress, malnutrition, or viral infection during pregnancy is
associated with an increased incidence of SZ later in life (Brown
et al., 1996; Brown, 2011; Koenig et al., 2002; Mednick et al.,
* Corresponding author. University of Illinois at Chicago (UIC), Department of
Psychiatry, 1601 West Taylor Street, Chicago, IL 60612, USA. Tel.: þ1 312 355 2438.
E-mail address: firstname.lastname@example.org (F. Matrisciano).
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ e see front matter Published by Elsevier Ltd.
Neuropharmacology 68 (2013) 184e194
1994; Izumoto et al., 1999; Susser et al., 1996). Animal studies
confirm the sensitivity of the developing brain to environmental
insults. For example, exposure of rats or mice to stresses, immune
challenges, infections and malnutrition during pregnancy leads to
disruption of behavioral and neurochemical parameters in adult
offspring that mimic aspects of major neuropsychiatric disorders
(Borrell et al., 2002; Moreno et al., 2011; Fatemi et al., 2008; Fortier
et al., 2007; Kinnunen et al., 2003; Koenig et al., 2005; Winter et al.,
2008; Zuckerman and Weiner, 2005; Shi et al., 2003). The above
studies and the lack of definitive genetic abnormalities causally
related to psychotic disorders suggest that the etiopathogenesis of
SZ and BP disorders may be at least in part epigenetic (Petronis
et al., 1999).
The epigenetic role of environmental factors in the pathogenesis
of SZ was suggested a decade ago in epidemiological studies by
Gottesman (1994). At the molecular level epigenetic studies in SZ
and BP disorder patients have focused on promoter cytosine
methylation, a covalent modification of DNA, in which a methyl
group is transferred from S-adenosyl-methionine (SAM) to the C-5
position of cytosine, by a family of DNA-methyltransferases (i.e.,
DNMT1, 3a, 3b, 3L). We and others have shown that DNMT1 and
DNMT3a are highly expressed in brain and these enzymes are
overexpressed in GABAergic neurons of SZ and BP disorder patients
(Veldic et al., 2005, 2007; Ruzicka et al., 2007; Guidotti et al., 2011).
Likely, as a consequence of the increased expression of DNA-
methyltransferases, reelin and other GABAergic or glutamatergic
gene promoters are hypermethylated and their expression is
downregulated in SZ (Veldic et al., 2005; Mill et al., 2008; Ruzicka
et al., 2007; Kundakovic et al., 2009; Grayson et al., 2005, 2006;
Zhang et al., 2010; Weaver, 2007; Sweatt, 2009).
Considering that high levels of DNMT are expressed in neuro-
progenitor cells and DNMT expression as well as DNA methylation
can be increased in post-mitotic neurons by early-life stressors
(Murgatroyd et al., 2009; Meaney and Ferguson-Smith, 2010;
Weaver et al., 2007), we propose that a putative mechanism by
which adverse prenatal experiences could provoke neuropsychi-
atric disturbances is bymodifying DNA methylation modulating the
expression of DNMT in GABAergic neurons. However, to our
knowledge, there is no evidence in mice regarding: (i) the
expression and function of DNMT in the developing brain, (ii)
DNMT changes in response to prenatal stress, or (iii) the effect of
epigenetic changes on behaviors related to SZ.
Here, we characterize a model of prenatal stress (PRS) in mice
seeking to establish whether restraint stress during pregnancy
results in an epigenetic GABAergic dysfunction that persists in the
brain of adult offspring and contributes to behaviors related to SZ
and/or other related neuropsychiatric disorders.
2.1. Prenatal stress paradigms
Pregnant Swiss- albino-ND4 mice (Harlan, Indianapolis) were individually
housed with a 12-h lightedark cycle and food and water ad libitum. Pregnant mice
were divided into two groups: one left undisturbed throughout gestation and one
subjected to a restraint stress using a plastic tube (10 cm ? 3 cm) for 30 min twice
daily from embryonic day 7e21 as described previously (Matrisciano et al., 2011).
Offspring not used for neurochemical measurements were weaned from their
natural mothers after 21 days; male PRS and control mice were housed separately (5
per cage), and left undisturbed for an additional 40e50 days until behavioral testing.
2.2. Quantitative competitive RT-PCR measurement of DNMT1 and DNMT3a mRNA
Total RNA was extracted from mouse frontal cortex (FC) (2 mm anterior to
bregma) and hippocampus with Trizol reagent (Invitrogen, Carlsbad, CA). The
absolute amount of DNMT1 and 3a mRNA was measured by RT-PCR using co-
linear internalstandards generatedby internal deletions.The internal
standards (IS) were designed by deleting 100- to 150-bp fragments from the
middle of the target cDNA sequence and were generated by an overlap extension
PCR with internal deletion primers (Auta et al., 2007). Amplification of cDNA was
carried out employing the following primers: DNMT1 e forward: 50TGA-
CAGTGGTGCTGAAGAAGCCAT 30, reverse: 50AGAATGGAGCCTCGAATTCTGAGA30;
DNMT3a e forward: 50AACAACAACTGCTGCAGGTGCTTT30, reverse: 50ACTCCTGGA
TATGCTTCTGTGTGA30. Concentrations of DNMT1 and DNMT3a mRNA were
calculated from a known amount of IS and corrected for nuclear specific enolase
(NSE) using the following primers e forward: 50AATCCAAGTTTGGGGCCAA
TGCCA30, reverse: 50TCTTTTCCGTGTAGCCAGCCTTGT30. The amplification prod-
ucts of IS and of the target gene were separated on agarose gels. The ratio of target
2.3. Western blot analysis
Western blot analysis was performed as described previously (Matrisciano et al.,
2002). Briefly, mouse FC was homogenized at 4?C in RIPA lysis buffer containing
1 mM of a protease inhibitor cocktail (Sigma), pH 7.4. Twenty micrograms of protein
were resuspended in a SDS-bromophenol blue reducing buffer. Western blot anal-
ysis was carried out using 4e12% tris-glycine gels (Invitrogen). After blotting onto
a nitrocellulose filter (0.2 mm pore size; Invitrogen), blots were incubated for 1 h at
room temperature with primary antibodies directed against DNMT1 (mouse
monoclonal; 0.5 mg/ml; Imagenex), reelin (1:5000, a generous gift of A.M. Goffinet,
University of Namur, Brussels), and GAD67 (mouse monoclonal, 1 mg/ml; Millipore)
in Tris-saline/Tween 20 buffer as described by Matrisciano et al. (2011). The same
blots were incubated for 1 h at room temperature with mouse monoclonal anti-
bodies directed againstb-actin (mouse monoclonal; 0.5mg/ml; Sigma) inTTBS buffer
(100 mM TriseHCl; 0.9% NaCl; 0.1% Tween 20; pH 7.4). After three washes with TTBS
buffer, blots were incubated for 1 h with peroxidase-conjugated secondary anti-
bodies (Sigma). Densitometric analysis was performed using a Storm 860 (Molecular
Dynamics, Sunnyvale, California) with IMAGEQUANT analysis software and the
values were expressed as an optical density (OD) ratio with respect to b-actin.
2.4. MeDIP and HMeDIP (methylated and hydroxymethylated DNA
immunoprecipitation) analysis of reelin and GAD67 promoters
We analyzed the ratio of 50methylated cytosines (5MC) or 50hydroxymethylated
cytosines (5HMC) to the unmethylated cytosines of mouse reelin (?432 to ?252) and
GAD67 (?840 to ?768) CpG-enriched promoter fragments. Mouse monoclonal 5MC
(Diagenode) or 5HMC (Active Motif) antibodies were used for the immunoprecipita-
tion. According to Gavin et al. (2012), we performed the MeDIP procedure because
bisulfite conversion and most enzyme-dependent methods are incapable of dis-
tinguishing 5MC from the approximately 20% of methylcytosines in the brain that are
Genomic DNA was extracted from the mouse FC and sonicated to produce a fragment
sizeof 200e600 bp. Afterethanolprecipitation, 3 mgof sonicated DNAwerediluted to
300 ml in TE buffer and heat-denatured at 95?C for 10 min. Then, 30 ml of sonicated
solutionwere removedandstoredat?20?Cto beusedtoquantifythe totalamountof
promoter before immunoprecipitation (input). The remaining solutionwas incubated
overnight at 4?C with mouse anti-5MC or anti-5HMC antibodies. The immunopre-
cipitated DNA was released from the antibody complex by proteinase-K digestion.
After phenol-chloroform extraction and ethanol precipitation, the DNA pellet was
resuspended in 20 ml of DEPC water. CpG-rich GAD67 and reelin gene promoters were
measured by Q-PCR. An immunoprecipitation negative control (no antibody added)
was included in each assay and did not produce any detectable signal.
2.5. ChIP assay: measurements of DNMT1 and methyl CpG binding protein 2
(MeCP2) binding to reelin and GAD67 promoters
We performed these experiments as previously described by Matrisciano et al.
(2011). Briefly, about 10 mg of tissue was used for this procedure. Tissue slices
were incubated with 500 ml of PBS containing 1% formaldehyde at 37?C for 10 min,
supplemented with a protease inhibitors cocktail (Sigma), and after three washes
with cold PBS, tissuewas homogenizedin 300ml of SDS lysis buffer (supplied byChIP
kit, Upstate). To obtain consistent chromatin fragmentation, the lysates were soni-
cated for 15 min on ice (Sonic Dismembrator, Model 500, Fisher Scientific). The ChIP
procedure was carried out by using the ChIP assay kit and protocol provided by the
manufacturer (Upstate, no. 17e295). The concentrations of DNMT1 (mouse mono-
clonal; Imagenex) and MeCP2 (rabbit polyclonal; Upstate) antibodies were 1 mg/ml.
An aliquot (2%) of the sonicated lysatewithout antibody (Input) was used to quantify
the total amount of DNA in sample extracts before immunoprecipitation. At the end
of ChIP procedure, the protein/DNA cross-linked nucleosomal chromatin complex
immunoprecipitated by specific antibody was reverse cross-linked with NaCl at
a final concentration of 100 mM at 65?C overnight. Samples were then treated with
proteinase-K.Protein-free DNAwas extracted inphenol/chloroform and precipitated
and washed in ethanol. The extract was used for detection and quantification of
reelin and GAD67 gene promoters.
F. Matrisciano et al. / Neuropharmacology 68 (2013) 184e194
CpG-rich reelin [(?432 to ?252 bp) (forward: GGGCGGCGGGCCCCGAGG,
reverse: AGAGACCGACGGGCTGCC)] and GAD67 [(from ?840 to ?768 bp) (forward:
GAGGAGAGCGGGCCAAGA, reverse: GTGCCGCTCCACACGCC)] promoter fragments
(see Fig. 4 for detailed sequences of the promoter regions) were measured by Q-PCR
as previously described (Matrisciano et al., 2011). The percent methylated vs.
unmethylated promoter was calculated by the following equation: % (meDNA-IP/
total input) ¼ 2^[(Ct(10% input) ? 3.32) ? Ct (meDNA ? IP)] ? 100% (MagMeDIP kit
instruction manual, Diagenode).
2.6. Confocal fluorescence microscopy in GAD67-GFP knock-in mice
To identify the location of DNMT with respect to neurons of defined neurotrans-
mitter phenotype, we used heterozygous knock-in enhanced green fluorescent
protein-glutamic acid decarboxylase 67 C57BL/6 mice (referred to as GAD67-GFP
mice), in which the cDNA encoding enhanced GFP is inserted into the GAD67 ATG
start codon by homologous recombination (Tamamaki et al., 2003). These mice were
obtained with permission of Dr. Yuchio Yanagawa, Department of Genetic and
Behavioral Neuroscience,11 Gunma University Graduate School of Medicine, Maeba-
shi 371e8511, Japan. Heterozygous GAD67-GFP breeder males were mated with wild
type C57BL/6 females and offspring were genotyped at birth. Two primer pairs were
used for genotyping so as to identify both alleles (GAD67-GFP and GAD67/GAD67) of
all offspring (Tamamaki et al., 2003). For the experiments described, 7 and 60-day old
animals were used. Coronal slices (nominally 20e25 mm thick) of fixed mouse brains
was performed as recently described (Kadriu et al., 2011). The following antibodies
were used: (i) polyclonal anti-GFP (diluted 1:250; Abcam; Cambridge, MA), (ii)
anti-DNMT3a (diluted 1:500; Imgenex, San Diego, CA).
To test the specificity of the immunological detection, the primary antibody was
omitted yielding no detectable fluorescent staining. Antibody specificity was eval-
uated by Western analyses of cortical extracts. Following separation on 4e20% SDS
polyacrylamide gels and blotting to nitrocellulose, major immunoreactive bands of
the expected molecular size were detected with all antibodies used. For more details
on the method see Kadriu et al. (2011).
2.7. Behavioral tests
Non-stressed (Control) and prenatally stressed (PRS) male mice at PND 60 were
used to examine the behavioral characteristics under basal conditions (drug- or
vehicle-free). For this purpose, we measured in order the 1) locomotor activity, 2)
social interaction with an intruder (unfamiliar, non-aggressive male mouse 60-days
old, Swiss albino-ND4 strain) in a novel environment, 3) prepulse inhibition at
startle, and 4) contextual fear conditioning. All behavioral tests were performed on
consecutive days. In the drug administration experiments, mice were injected with
valproic acid (70 mg/kg, i.p.; twice a day for 5 days), or clozapine (5 mg/kg, s.c.;
twice a day, for 5 days), or vehicle and then, 24 h after the last drug injection,
animals were tested for locomotor activity, social interaction, PPI, and fear condi-
tioning over the next four days with drugs being administered after completion of
each daily test. In another set of experiments, control and PRS mice were injected
with clozapine (5 mg/kg, s.c., twice a day) or vehicle for five days and then with
a single injection of MK-801 (0.1 mg/kg s.c.) or vehicle 30 min before the test. All
animals were housed in the experimental room an hour prior to the test session for
2.7.1. Locomotor activity
A computerized Animal Activity Monitoring System with VersaMax software
(AccuScan Instruments, Columbus, Ohio) was used for the quantification and
tracking of locomotor activity in mice as described previously (Carboni et al., 2004).
Each activity cage consisted of a Perspex box (20 ? 20 ? 20 cm) surrounded by
horizontal and vertical infrared sensor beams. The total number of interruptions of
the horizontal sensors was taken as a measure of horizontal activity whereas that of
vertical sensorswas used as a measureof verticalactivity. Thepercentageof distance
traveled in the center of the field with respect to the margins was also calculated.
Activity was recorded for 20 min.
2.7.2. Stereotypic behavior
Stereotypy was measured using AccuScan software. Repeated beam breaks on
the same beam (or set of beams) were recorded as an index of stereotypic activity.
Activity was recorded for 20 min.
2.7.3. Social interaction in a novel environment
We used the experimental paradigm described by Tremolizzo et al. (2005). In
brief, individual mice were placed in a novel cage together with an unfamiliar, non-
aggressive 60-day old male mouse of the same strain used as an intruder and the
interactions between the two mice (initiated by the experimental mouse) were
recorded for 10 min with a digital webcam (Logitech, Inc, California, USA). The time
spent in social interaction (sec/10 min) was scored by two well-trained blind
operators. Social interaction was defined by body contact including inspection and
Fig.1. Prenatal stress (PRS) causes an early and long lasting increase in the expression of DNMT1 and 3a in the mouse frontal cortex and hippocampus. DNMT1 and 3a mRNA levels
in the frontal cortex (FC) of control (Ctrl) and PRS mice at PND 1, 7,14, and 60 are shown in (A); levels in the hippocampus of Ctrl and PRS mice at PND 7 and 60 are shown in (B). NSE
and GPDH were utilized as internal controls. All values (A, B) are means ? S.E.M. of six mice. *p < 0.05. (Student’s t test) vs. the corresponding Ctrl value.
F. Matrisciano et al. / Neuropharmacology 68 (2013) 184e194
ano-genital sniffing. Reliability of measurements was assessed by correlating the
scores of two raters.
2.7.4. Prepulse inhibition of startle (PPI)
Startle was recorded using the SR-Lab Startle Response System (San Diego, Cal-
ifornia). The startle box was programmed to record five 120 db startle pulses
(broadband noise 30 msec in duration), at the beginning and at the end of the 20 min
session. The PPI trial sequence consisted of the presentation of 50 pseudo-random
trials, 10 for each of the following trial types: (i) no stimulus; (ii) a startle-only pulse
at 120 db, and prepulse-startle trials with a (iii) 74 db-, (iv) 78 db-, or (v) 82 db-
was 100 msec. Startle amplitude was defined as the maximum amplitude within
a 100 msec windowfollowing the presentation of the startle pulse. Intertrial intervals
were random (mean ITI ¼ 15 s). The amount of inhibition was calculated as the
following ratio: mean startle for startle-only trials minus mean startle for prepulse-
trials divided by mean startle for startle-only trials multiplied by 100.
2.7.5. Contextual fear conditioning
We used the experimental paradigm described by Pibiri et al. (2008). The fear-
conditioning apparatus consisted of a transparent acrylic chamber measuring 25 cm
Fig. 2. DNMT1 is co-expressed with GFP in cortico-limbic GABAergic neurons of the GAD67-GFP knock-in mouse. Confocal double immunofluorescence labeling of GFP (green),
DNMT1 (red), and merged images in orange (center panels). A) AeC: motor cortex layer II of a 7-day old PRS mouse; DeF: motor cortex layer II of a 7-day old control mouse. Coronal
sections correspond roughly to bregma ?1.4 mm. B) AeC: CA1 field of the hippocampus of the PRS mouse; DeF: CA1 field of the hippocampus of the control mouse. Coronal
sections correspond roughly to bregma ?2 mm. Scale bars for all panels represent 20 mm.
F. Matrisciano et al. / Neuropharmacology 68 (2013) 184e194
wide, 18 cm high, and 21 cm deep (San Diego Instrument, Inc., San Diego, CA). The
cagefloorconsistedof stainless steel rodsconnectedtoan electric shockgenerator. A
small fan was located on the top wall of the enclosure. A speaker placed on a side
wall of the conditioning chamber delivered the auditory tone. The chamber was
surrounded bya frame with 16 infrared photo beams. Computer software controlled
the delivery of electric foot shocks and auditory stimuli and recorded beam inter-
ruptions and latencies to beam interruptions (freezing time).
Training session: mice were placed into the chamber and allowed to explore it
for 2 min. After this time, they received an acoustic tone (conditioned stimulus, CS)
(30s, 85 DB) co-terminated with an unconditioned stimulus (US) (electric footshock,
Fig. 3. PRS induced changes in the DNMT1, reelin, and GAD67 protein levels in the mouse frontal cortex. Immunoblot analysis shows an increase in the protein levels of DNMT1, and
a marked decrease in reelin and GAD67 protein levels in 60-day old PRS mice compared to controls. The representative immunoblots show a major band of 190 kDa for DNMT1,
160 kDa for reelin, and 67 kDa for GAD67. All values are means ? S.E.M. of five mice. *p < 0.05 (Student’s t test) vs. the corresponding Ctrl value. All data were normalized by b-actin
Fig. 4. PRS causes an increase of: A) DNMT1 binding to specific reelin (?432 to ?252) and GAD67 (?154 to þ21) promoter regions in the FC of PND 60 mice; B) MeCP2 binding to the
same reelin and GAD67 promoter regions in the FC of PND1 and PND 60 mice. C) 5-methylcytosine binding at reelin and GAD67 promoters in the FC of PND 60 mice, and D) 5-
hydroxymethylcytosine binding at reelin and GAD67 promoters in the FC of PND 60 mice. Values are means ? S.E.M. of 6 mice. p < 0.05 (Student’s t test) vs. the corresponding
control (Ctrl) values (*) or vs. saline (#).
F. Matrisciano et al. / Neuropharmacology 68 (2013) 184e194
2 s, 0.5 mA). The tone plus the foot shock were repeated for three times every 2 min.
After the last tone þ shock delivery, mice were allowed to explore the context for an
additional minute prior to removal from the training chamber.
Test session: 24 h after training, mice were placed in the same chamber and
freezing behavior was measured for 5 min without tone or footshock presentation.
Freezing was defined by the absence of any movement except for those related to
respiration while the animal was in a stereotyped crouching posture. To measure
extinction, mice were re-exposed over the next nine days to the conditional context
without receiving footshock or acoustic tone.
2.8. Statistical analysis
Results are expressed as mean ? SEM. Experimental differences were assessed
by Student’s t test, one way ANOVA followed by multiple comparisons with the
Bonferroni test, or two way repeated measures ANOVA followed by multiple
comparisons (Bonferroni or Student-NewmaneKeuls comparison) as indicated in
figure legends. The criterion for significance was p < 0.05, two tailed. SigmaStat 2.03
statistical software (SPSS inc.) was used for the analysis.
3.1. Increased expression of DNMT1 and 3a in frontal cortex (FC)
and hippocampus of PRS mice
offspring born from non-stressed mothers (control mice) at post-
considerably higher than DNMT3a mRNA levels, the expression of
both was markedly elevated at PND1 but then decreased dramati-
cally and progressively up to PND 60 (Fig.1A). In the FC of offspring
born from stressed mothers (PRS mice), we found that DNMT1
(Fig. 1A top) and DNMT3a (Fig. 1A bottom) mRNA levels were
significantly higher at PND 1, 7,14, and 60, compared to controls.
We also measured the DNMT mRNA levels in the hippocampus
at PND 7 and 60.
As shown in Fig. 1B, PRS mice showed a marked increase in
DNMT1 (Top) and DNMT3a (bottom) mRNA levels compared to
control mice. As in the FC, DNMT1 mRNA levels were higher than
DNMT3a mRNA levels in the hippocampus (Fig. 1B).
3.2. DNMT is primarily expressed in GABAergic interneurons
We have previously reported that DNMT1 and 3a are highly
localized in GABAergic neurons in the cortex and hippocampus of
adult human and mouse brains (Veldic et al., 2005; Ruzicka et al.,
2007; Satta et al., 2008; Kadriu et al., 2011). Here, using GAD67/
GFP knock-in mice that are extremely sensitive to stress exposure
(Uchida et al., 2011), we confirmed the almost complete co-
localization of GAD67 and DNMT1 protein in cortical and hippo-
campal neurons of 7-day old control and PRS mice (Fig. 2). Similar
results were obtained for DNMT3a (data not shown). Hence, the
higher levels of DNMT in the cortex and hippocampus of 7-day old
control and PRS mice as compared to the respective 60-day old
mice, presumably reflect the presence in brain of GABAergic
neurons overexpressing DNMT.
3.3. Altered expression of schizophrenia-related genes in the frontal
cortex of PRS mice
Earlier reports suggest that an increase in DNMT levels is
associated with a downregulation of the gene encoding the GABA-
synthesizing enzyme GAD67, or the trophic protein reelin in post-
mortem brain tissue from patients affected by SZ or BP disorders
(Guidotti et al., 2011) and in brain of rats exposed to early-life post-
natal stress (Zhang et al., 2010). Here, we report that, associated
with an increased level of DNMT, 60-day old PRS mice show
a marked decrease in GAD67 and reelin protein levels in the FC
(Fig. 3), when compared to control mice.
3.4. DNMT binding to GABAergic gene promoters
To test whether the overexpression of DNMT1 in FC of PRS mice
correlates with an increased binding of DNMT1 to specific reelin
and GAD67 CpG-rich promoter sequences, we measured the
binding of DNMT1 to these promoters by ChIP assay. We showed
that in the FC of 60-day old PRS mice, the binding of DNMT1 to the
same reelin (?432 to ?252); and GAD67 (?154 to þ21) promoter
regions was increased (Fig. 4A).
To study whether the bindingof DNMT1 correlateswith changes
in the methylation of these promoters we assessed: a) binding of
MeCP2 to specific CpG-rich reelin and GAD67 promoter regions, and
b) MeDIP and HMeDIP measurements of reelin and GAD67
As shown in Fig. 4B, at PND1, when the levels of DNMT were
highest, and at PND 60 when the levels of DNMT were lowest, the
binding of MeCP2 to specific reelin and GAD67 promoter regions
was highest at day 1 and markedly decreased at day 60.
Moreover, the MeCP2 binding to GAD67 and reelin gene
promoters was significantly higher in PRS mice (Fig. 4B). We
recentlyobserved that changes in MeCP2 binding in PRS mice occur
in the absence of changes in MeCP2 levels (Matrisciano et al., 2011).
In Fig. 4C and D we show that the 5MC and 5HMC covalent
modifications at the reelin and GAD67 promoters were increased in
60-day old PRS mice compared to controls.
3.5. PRS mice show a schizophrenia-like behavioral phenotype
We made the consistent observation that PRS mice were born
12e18 h before control mice and had lower (10e15%) body weight
at birth (control mice weight: 4.06 ? 0.4 g) as previously reported
by Torche and Kleinhaus (2011). However, PRS mice had no obvious
differences in body weight, auditory sensitivity, pain sensitivity, or
motor coordination when compared with offspring born from
unstressed mothers at PND 60. In contrast, offspring of stressed
pregnant mothers at 2e3 month of age (late adolescence/early
adulthood in mice) demonstrate alterations in behavioral tests.
3.5.1. Spontaneous locomotor activity
In Fig. 5A, we report that 60e70-day old PRS male mice showed
a robust and persistent hyperactivity as measured by horizontal
activity in an open field arena over the 20 min of the test. These
mice were also characterized by an increase in the percentage of
distance traveled in the center of the field with respect to the
3.5.2. Social interaction
Social withdrawal is a frequent negative symptom of SZ. In mice,
social withdrawal can be evaluated using the social interaction test
(Tremolizzo et al., 2005). The same PRS mice subjected to the social
interaction test showed a significant decrease in social interactions
with an unfamiliar mouse during a 10-min test period compared to
controls (Fig. 5B).
3.5.3. Deficits in attention and information processing
Deficits in information processing and attention have been
considered central features of SZ which may lead to stimulus
overload, cognitive fragmentation, and thought disorders.
A well-established method for evaluating the status of infor-
mation processing across species including humans and mice is
prepulse inhibition of startle (PPI). Our 60e70-day old PRS mice
showed a significant PPI deficit (Fig. 5C; 2-way repeated measure
ANOVA followed by Bonferroni comparison), a condition that is
similar to the deficit in sensory-motor gating seen in SZ patients
(reviewed by Braff et al., 2001).
F. Matrisciano et al. / Neuropharmacology 68 (2013) 184e194
Another paradigm useful for evaluating the integrity of learning
and memory processes in cortico-limbic structures is fear condi-
tioning. In our experiments normal mice exhibited freezing
behavior if re-exposed after 24 h to the context in which they had
previously received an electric shock. This freezing response
extinguished with repeated re-exposure to the context without
footshock or acoustic tone (Fig. 5D). PRS mice demonstrated
a significant decrease in fear conditioning response and no
extinction (Fig. 5D; 2-way repeated measure ANOVA followed by
Bonferroni comparison) which may be reminiscent of abnormal
cognitive processing in SZ (Amann et al., 2010). There was no
difference in freezing behavior measured during the habituation
phase or during the training session suggesting that PRS mice fail to
exhibit alterations in unconditioned fear-related behavior, pain or
3.5.4. Stereotypic behavior
PRS mice show an increase in stereotypy when compared to
control mice. PRS mice responded to low doses (0.1 mg/kg s.c.) of
the NMDA receptor antagonist MK-801 with a further increase in
stereotypy whereas control mice failed to respond to this low dose
of MK-801 (Fig. 6).
3.6. Valproate (VPA) and clozapine correct SZ-like behavior in PRS
To test whether the behavioral alterations of offspring of PRS
mice were mediated by epigenetic mechanisms including an
increase in DNMTs, an increase of GABAergic promoter methyla-
tion, and a downregulation of the GABAergic gene expression, we
evaluated the behavior of these mice following repeated treatment
with the histone deacetylase (HDAC) inhibitor VPA and the chro-
matin remodeling antipsychotic drug clozapine (Guidotti et al.,
2011). We have previously reported that VPA, which is known to
induce promoter demethylation by activating DNA-demethylation
mechanisms (Szyf, 2011; Guidotti et al., 2009, 2011), corrects the
reelin and GAD67 promoter hypermethylation, the decrease of
GAD67 and reelin expression, and the PPI and social interaction
deficits induced by protracted (one week) methionine treatment
(Tremolizzo et al., 2005). Here, we show that VPA corrected the
hyperactivity (Fig. 7A), the social interaction (Fig. 7B), and PPI
Fig. 6. Increased stereotypic behavior in PRS mice induced by NMDA receptor
blockade with a low dose of MK-801. Stereotypy counts are shown in control and PRS
mice pretreated with clozapine (5 mg/kg s.c. twice a day for 5 days) and given a single
s.c. injection of MK-801 (0.1 mg/kg) 24 h after the last injection of clozapine and
30 min before the behavioral test. Values are means and ?S.E.M. Two way repeated
measures ANOVA: Group effect (F3,18¼ 5.736, p ¼ 0.006; MK-801 effect (F1,18¼ 5.939,
p ¼ 0.025); Group ? MK-801 interaction (F3,18¼ 3.198, p ¼ 0.48). NewmaneKeuls post-
hoc comparisons: vs. control mice treated with vehicle (*) or vs. PRS mice treated with
Fig. 5. PRS is associated with schizophrenia-like behavioral abnormalities. Sixty-day old PRS mice compared to controls were characterized by: (A) an increase in locomotor activity
t23¼ 2.466, p ¼ 0.02); (B) a decrease in social interaction (t16¼ 3.455, p < 0.001); (C) a deficit in prepulse inhibition of startle (2-factor ANOVA: F1,16for group ¼ 14.826, p ¼ 0.001,
F2,16for prepulse intensity ¼ 12.503, p < 0.001, group by intensity interaction not significant; and (D) a deficit in contextual fear conditioning (2-factor ANOVA on freezing on re-
exposure to the context over the first three days following conditioning F1,9¼ 6.735, p ¼ 0.029); Bonferroni comparison: t(10)2.59, p ¼ 0.029 Further, regression analysis revealed
that contextual fear extinguished in controls to PRS level within 9 days following conditioning (F1,9¼ 5.334, p ¼ 0.002).
F. Matrisciano et al. / Neuropharmacology 68 (2013) 184e194
(Fig. 7C) deficits in offspring of prenatally stressed mothers at doses
that had no major effect in control mice.
Clozapine, which in clinically relevant doses also induces chro-
matin remodeling and facilitates DNA promoter demethylation,
(Guidotti et al., 2009) also corrected the behavioral abnormalities
observed in adult PRS mice (Fig. 7AeC). In addition, a low dose
(0.1 mg/kg) of the NMDA receptor antagonist, MK-801, significantly
increased stereotypic behavior in vehicle-treated PRS mice but not
in clozapine pretreated PRS mice or in controls (Fig. 6).
Several lines of evidence point to SZ as a neurodevelopmental
disorder inwhich stress or environmental insults during pregnancy
or in early-life contribute to the onset of the disease by altering
epigenetic DNA marking preferentially at cortical and hippocampal
GABAergic neurons (Zhang et al., 2010; Guidotti et al., 2011; Benes,
2011; Grayson, 2010; Brown, 2011; Fatemi et al., 2008; Markham
and Koenig, 2011; Howes et al., 2004).
In rats, exposure to stress during gestation induces marked
changes in the behavior of the offspring that are reminiscent of the
positive, negative and cognitive symptoms present in SZ and BP
disorder patients (Lemaire et al., 2000; Koenig et al., 2005). Our
results provide further evidence that restraint stress during preg-
nancy in mice leads to a clozapine- and VPA-sensitive behavioral
psychosis-like phenotype in offspring (PRS mice) strongly sug-
gesting that PRS mice represent a valid behavioral animal model of
SZ and BP disorders.
Here, we demonstrate for the first time that, in PRS mice,
“psychotic-like” behavior (hyperactivity, enhanced responsetoMK-
801, and a deficit in social interaction, PPI, and fear conditioning)
measured in the adult is associated with a persistent upregulation
of DNMT1 and 3a in cortical and hippocampal GABAergic neurons
during neurodevelopment and by a sustained binding of DNMT to
GABAergic gene promoters. These epigenetic events are associated
with hypermethylation of reelin, GAD67, and likely other GABAer-
gic promoters (as determined by the MeDIP, HMeDIP assays and by
the increase in MeCP2 binding) and with a persistent down-
regulation of the expression of the respective genes. Importantly,
the epigenetic phenotype present in cortex and hippocampus of
PRS mice is reminiscent of the epigenetic phenotype shown in the
brain of patients with SZ and BP disorders (Guidotti et al., 2011;
Roth et al., 2009). When the post-mortem brains of SZ and BP
disorder patients are compared with that of non psychotic subjects,
a GABAergic neuropathology is detected in the prefrontal cortex
and hippocampus (Guidotti et al., 2005). This GABAergic neuropa-
thology is not associated with neuronal loss but is characterized by
decreased 1) GAD67, the rate limiting step enzyme in GABA
synthesis (Akbarian et al.,1995; Volk et al., 2000; Benes et al., 2007;
Huang and Akbarian, 2007; Guidotti et al., 2000); 2) reelin, the
extracellular matrix protein that regulates dendritic spine matu-
ration and glutamate receptor structure and function (Guidotti
et al., 2000; Fatemi et al., 2000); and 3) other genes encoding
markers of GABAergic interneurons, including the NMDA receptor
subunit NR2A (Woo et al., 2008), the nAChR subunits a4, b2, and a7
(Breese et al., 2000), the high affinity GABA transporter, somato-
statin, and cholecystokinin (Lewis et al., 2005; Benes et al., 2007).
We have recently shown that DNMT is highly expressed in
telencephalic GABAergic neurons of the adult mammalian brain
and is preferentially overexpressed in GABAergic neurons of SZ and
Fig. 7. Schizophrenia-like behavioral abnormalities in PRS mice are reversed by treatment with valproic acid (70 mg/kg, i.p.; twice a day for 5 days), or clozapine (5 mg/kg, s.c.; twice
a day, for 5 days). Data of locomotor activity (A), social interaction (B), and PPI (C), 24 h after the last drug injection, are shown. One way ANOVA comparing controls (NS) veh, PRS
veh, PRS VPA and PRS clozapine: (A) locomotor activity (F3,33¼ 8.919, p ¼ 0.001), (B) social interaction (F3,28¼ 17.233, p < 0.001, and (C) PPI (74 db F3,42¼ 5.189, p ¼ 0.004; 78 db
F3,42¼ 9.625, p < 0.001; 82 db F3,42¼ 6.582, p < 0.001). Post-hoc NewmaneKeuls comparisons indicated that PRS veh mice differed from controls veh mice (*), and from PRS mice
treated with VPA (**) or clozapine (#). Analysis for PRS mice considered yielded a similar result while ANOVAs for controls were not significant.
F. Matrisciano et al. / Neuropharmacology 68 (2013) 184e194
BP disorder patients (Veldic et al., 2007; Zhubi et al., 2009; Ruzicka
et al., 2007; Kadriu et al., 2011). Comparingthe expression of DNMT,
reelin, GAD67 and other GABAergic genes in laser microdissected
GABAergic interneurons and pyramidal neurons of SZ patients, we
were able to establish that the overexpression of DNMT and the
downregulation of GABAergic genes in GABAergic interneurons are
closely related events (Ruzicka et al., 2007).
It is plausible that in the human and mouse brain, DNMT-
induced GABAergic deficits could be the basis for the disturbance
of the reciprocal interaction between GABAergic, glutamatergic and
monoaminergic neurons that likely represents the mechanism
underlying the exacerbation of psychotic episodes elicited by
NMDA receptorantagonists to SZ and BP disorder patients. (Meltzer
et al., 2011; Javitt, 2007; Breese et al., 2002; Lisman et al., 2008).
may also explain the stereotypic hypersensitivity to administration
of low doses of the NMDA receptor antagonist MK-801 in PRS mice
(see Fig. 6).
In view of the evidence that alterations in DNA methylation are
involved in the etiopathogenesisof SZ (Mill et al., 2008; Veldic et al.,
2005; Grayson et al., 2006; Tremolizzo et al., 2005) and that high
levels of DNMT are expressed in the developing brain (Fig. 1)
(Guidotti et al., 2011; Zhang et al., 2010; Weaver et al., 2007),
a putative mechanism bywhichadverse prenatal experiences could
perturb GABA-glutamate interactions and exacerbate the stereo-
typic behavior induced by the NMDA receptor antagonist MK-801
may involve alterations in DNA methylation and the expression of
DNMT in cortico-limbic GABAergic neurons.
To further investigate the hypothesis that prenatal stress may be
responsible for the epigenetic alterations of GABA-glutamate
neuron interactions in PRS mice, we administered VPA and cloza-
pine in doses that are known to act on chromatin remodeling
inducing reelin and GAD67 promoter demethylation (Guidotti et al.,
2009). Under these conditions, VPA and clozapine abolished the
hyperactivity, stereotypy, social interaction, and PPI shown in PRS
mice whereas no effects were observed in control mice. Further-
more, clozapine blocked the increased stereotyped behavior in PRS
mice induced by low doses of MK-801. It is noteworthy that the
doses of VPA and clozapine active on behavior in PRS mice fail to
have a significant effect on the behavior of control mice, suggesting
a specificity of action on the epigenetic mechanisms that underlie
the behavioral pathology in PRS mice. We are now testing this
PRS mice and post-mortem brain of SZ patients have in common
increased levels of DNMT. From the data presented in Fig. 1, which
show higher levels of DNMT in cortex and hippocampus from birth
to adulthood, we can infer that in PRS mice the increase in DNMT is
probably the result of changes occurring during embryonic life. The
importance of stress during the embryonic period is indicated by
our finding that DNMT was elevated in PRS mice even on Day 1
before differences in maternal care could be a significant factor.
Further, it has been reported in Swiss mice that stressed mothers
raising stressed pups exhibit maternal care comparable to that of
non-stressed mothers raising non-stressed pups (Meek et al.,
We cannot establish at the present time if a similar time course
in changes in DNMT occur in the human brain but it is conceivable
that similar neurodevelopmental changes can occur as a response
to stressful situations either in utero or during early post-natal life,
thus preventing the reduction in DNMTexpression that occurs with
development. This hypothesis is supported by reports that as in our
mouse model, the exposure of pregnant women to psychological
stress, malnutrition, or viral infection during pregnancy is associ-
ated with an increased incidence of psychosis later in the life of
offspring (Mittal et al., 2008; Markham and Koenig, 2011; Howes
et al., 2004). Hence, the neurochemical, behavioral, and pharma-
cological responses observed in adult offspring of mothers exposed
to stress during pregnancy appear to parallel some of the responses
obtained in the adult onset of SZ and strongly support the prenatal
stress model in mice as a pertinent endophenotypic epigenetic
animal model of psychosis.
Taken together, the effects of prenatal stress on behavior are
indicative of neocortical inhibitory/excitatory circuit imbalance and
changes in the expression of DNMT, GAD67, and reelin, suggesting
that PRS mice can be a valid animal model to study the epigenetic
mechanisms underlying SZ and BP disorders. PRS mice are suitable
for validating new compounds with potential antipsychotic activity
acting at an epigenetic level as shown for mGlu2/3 metabotropic
glutamate receptor agonists (Matrisciano et al., 2011).
Disclosure/conflict of interest
All Authors (FM, PT, ID, BK, DG, FN, GA) deny any conflict of
interest for the preparation of this paper.
This work was supported in part by the National Institutes of
Health, National Institute of Mental Health (Grant MH0708551
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