Elsevier

Neuropharmacology

Volume 68, May 2013, Pages 184-194
Neuropharmacology

Epigenetic modifications of GABAergic interneurons are associated with the schizophrenia-like phenotype induced by prenatal stress in mice

https://doi.org/10.1016/j.neuropharm.2012.04.013Get rights and content

Abstract

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 methylation.

This article is part of the Special Issue entitled ‘Neurodevelopmental Disorders’.

Highlights

► Prenatal restraint stress (PRS) in mice as a model of schizophrenia. ► Epigenetic alterations of DNMT1 and 3a, reelin and GAD67 in PRS mice. ► DNMT1 and 3a were preferentially expressed in GABAergic interneurons. ► Schizophrenia-like behavioral abnormalities in adult PRS mice.

Introduction

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 neurodevelopmental 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 psychological 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., 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 neuroprogenitor 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 neuropsychiatric disturbances is by modifying 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.

Section snippets

Prenatal stress paradigms

Pregnant Swiss- albino-ND4 mice (Harlan, Indianapolis) were individually housed with a 12-h light–dark 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 7–21 as described previously (Matrisciano et al., 2011). Offspring not used for neurochemical measurements were weaned from their natural mothers after 21

Increased expression of DNMT1 and 3a in frontal cortex (FC) and hippocampus of PRS mice

We first measured DNMT1 and DNMT3a mRNA levels in the FC of offspring born from non-stressed mothers (control mice) at post-natal days (PND) 1, 7, 14 and 60. Although, DNMT1 mRNA levels were considerably higher than DNMT3a mRNA levels, the expression of both was markedly elevated at PND1 but then decreased dramatically 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

Discussion

Several lines of evidence point to SZ as a neurodevelopmental disorder in which 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

Conclusions

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 (

Disclosure/conflict of interest

All Authors (FM, PT, ID, BK, DG, FN, GA) deny any conflict of interest for the preparation of this paper.

Acknowledgment

This work was supported in part by the National Institutes of Health, National Institute of Mental Health (Grant MH0708551 to AG).

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