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Early life stress (ELS) programs the developing organism and influences the development of brain and behavior. We tested the hypothesis that ELS-induced histone acetylations might alter the expression of synaptic plasticity genes that are critically involved in the establishment of limbic brain circuits. Maternal separation (MS) from postnatal day 14–16 was applied as ELS and two immediate early genes underlying experience-induced synaptic plasticity, Arc and early growth response 1 (Egr1) were analyzed. We show here that repeated ELS induces a rapid increase of Arc and Egr1 in the mouse hippocampus. Furthermore, immunoblotting revealed that these changes are paralleled by histone modifications, reflected by increased acetylation levels of H3 and H4. Most importantly, using native Chromatin immunoprecipitation quantitative PCR (nChIP-qPCR), we show for the first time a correlation between elevated histone acetylation and increased Arc and Egr1 expression in response to ELS. These rapid epigenetic changes are paralleled by increases of dendritic complexity and spine number of hippocampal CA3 pyramidal neurons in ELS animals at weaning age. Our results are in line with our working hypothesis that ELS induces activation of synaptic plasticity genes, mediated by epigenetic mechanisms. These events are assumed to represent early steps in the adaption of neuronal networks to a stressful environment.
The majority of epidemiological and experimental studies have focused on the detrimental consequences of early life stress (ELS) for the development of emotionality and cognition, and have emphasized that ELS imposes an elevated risk for depression, anxiety disorders, and substance abuse later in life (Heim and Nemeroff 2001; Keyes et al. 2011; McClelland et al. 2011; Schmidt et al. 2011). Correlated with the emotional and cognitive behavioral deficits, ELS elicits long-lasting changes in synaptic wiring of prefronto-limbic circuits. For instance, a number of studies have shown that pre- and early post-natal stress experience results in significant structural changes in the prefrontal cortex and limbic brain regions such as the hippocampus, including alterations of dendrite arborisation and the density of dendritic spines in late life (Bock et al. 2005a, 2011; Murmu et al. 2006; Kolb et al. 2012; McEwen et al. 2012).
On the other hand, evidence is accumulating that ELS induces sustained epigenetic changes (i.e., alterations in genomic expression that occur independent of changes in gene sequence), including DNA methylation and histone modifications (Weaver et al. 2004; Roth and Sweatt 2011; Levine et al. 2012; McEwen et al. 2012). There is also increasing evidence suggesting that epigenetic programming plays an important role in experience-induced synaptic plasticity (Gräff and Mansuy 2008; Fagiolini et al. 2009; Day and Sweatt 2011; McClelland et al. 2011).
According to Meaney and Ferguson-Smith (2010) ‘Epigenetic states lie at the interface between environmental signals and genome, serving to govern dynamic changes in transcriptional activity through extra- and intracellular mediators. In a multistep process, the epigenetic template attracts specific effectors that determine the responsivity of specific genomic regions to environmentally induced intracellular signaling pathways, thus leading to more stable effects on the potential for transcriptional activation and variation in neural function’. It is important to note that the establishment of a stable inherited epigenetic state cannot occur without the first, dynamic step (Dudley et al. 2011). So far, most studies focus on long-term epigenetic changes after stress and their associations with synaptic structure and behavior, whereas much less is known about ELS-induced early changes in gene expression and their regulation by epigenetic mechanisms, which represent the first step in the above mentioned concept. Thus, the aim of this study was to test our working hypothesis that ELS induces rapid alterations in the acetylation of histones H3 and H4, which correlate with the expression of Arc and Egr1, two key molecules underlying learning and experience-induced synaptic plasticity (Steward et al. 1998; Knapska and Kaczmarek 2004; Bock et al. 2005b; Thode et al. 2005; Tzingounis et al. 2006; Bramham et al. 2008; Korb and Finkbeiner 2011). We also analyzed in which way ELS results in neuromorphological alterations in hippocampal neurons (CA3 region and dentate granule cells) at weaning age, similar to findings in the prefrontal cortex (Bock et al. 2005a).
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This study is to our knowledge the first to show that ELS, that is, repeated periods of MS during early childhood, induces a rapid increase in the plasticity-related genes Arc and Egr1 in the mouse hippocampus. Furthermore, we found that these changes in gene expression are paralleled by histone modifications, reflected by increased acetylation levels of H3 and H4. Most importantly, our results reveal for the first time a correlation between elevated histone acetylation and increased Arc and Egr1 expression in response to ELS. The epigenetic changes observed at the age of PND 16 are paralleled by increased dendritic spine numbers, longer and more complex dendrites on CA3 pyramidal neurons in the MS group at weaning age compared to controls.
As opposed to adult stress (Hinwood et al. 2011; Castellano et al. 2012; Hollis et al. 2012) and DNA methylation (Murgatroyd et al. 2009; Franklin et al. 2010), only few studies have identified changes in histone acetylation after ELS (Levine et al. 2012). The rise in corticosterone after the last stress episode in our study indicates an endocrine stress response to this unpredictable and unavoidable stressful situation. Therefore, the elevations of gene expression are presumably mediated by stress-induced histone modifications, since both, the levels of acetylated H3 and of H4 were increased in the hippocampus of MS animals. Evidence for a direct correlation between the increase in stress hormones and enhanced histone acetylation comes from a study of Roozendaal and colleagues, where it was shown that the systemic application of corticosterone increases histone acetylation in the insular cortex and hippocampus (Roozendaal et al. 2010).
More importantly, only few studies on ELS have analyzed correlations between rapid histone modifications and the regulation of specific genes, related to synaptic plasticity. ChIP-qPCR analysis revealed that only the enhanced acetylation of H4 was associated with the promoter regions of Arc and Egr1, indicating that the MS-induced increase in H4 acetylation up-regulates Arc and Egr1 gene expression in the hippocampus.
Most studies that applied MS as a strong emotional stimulus for exploring the etiology and vulnerability of affective disorders applied chronic repeated (over 14 days) or extended (24 h) MS paradigms (Heim and Nemeroff 2001; Monroy et al. 2010; Schmidt et al. 2011; Levine et al. 2012). In contrast to that, the epigenetic and structural changes observed in our study were the result of only three brief (3 h) episodes of ELS. Therefore, these findings reflect rapid (30 min after stress exposure) epigenetic alterations in response to an environmental challenge, which were found for both the Arc and Egr1 promoters. It is important to note that this study was hypothesis driven and not aimed at screening for all genes that might be altered in response to ELS. Thus, we focused on the two synaptic plasticity genes Arc and Egr1, but we cannot rule out alterations of other genes.
Egr1 and Arc are well characterized activity-regulated immediate early genes which play important roles in synaptic plasticity and memory functions (Lyford et al. 1995; Knapska and Kaczmarek 2004; Bramham et al. 2008; Davis et al. 2010; Korb and Finkbeiner 2011). For Egr1, several potential target genes have been described (James et al. 2005) and in this context it is of particular interest that Arc was identified as a direct target of Egr1, as it was shown that Egr1 binds to the Arc promoter (Li et al. 2005). Therefore, it is tempting to speculate that ELS induces an increase in Egr1 expression regulated by histone acetylation, which then in turn up-regulates the expression of Arc. Arc is a direct effector protein in dendrites and at the synapse (Steward et al. 1998). In addition, Arc mRNA is translocated to dendrites, accumulates at active synaptic sites where it is locally translated and thereby interferes with dendritic and synaptic growth and reorganization (Bramham et al. 2008; Korb and Finkbeiner 2011). In addition to histone acetylation, the expression of Arc is also regulated as a late-response gene via protein synthesis-dependent mechanisms (Li et al. 2005). These changes might be mediated by changes in stress hormones during ELS exposure. The rise in corticosterone after the last stress episode indicates an endocrine stress response. Regulation of Arc expression via stress hormone receptors was recently demonstrated in the hippocampus of glucocorticoid receptor (GR)(+/−) mice, where the reduction in GR protein was accompanied by a decrease in Arc protein (Molteni et al. 2010). In line with this finding, a recent study showed that Arc expression in the hippocampus can be activated via GR's and the CaMKIIα-BDNF-CREB pathway, mechanisms that are critically involved in the regulating of memory consolidation (Chen et al. 2012). Similarly, there is evidence that stress-induced activation of GR also regulates Egr1 expression (Revest et al. 2005).
We speculate that the observed histone modifications together with the regulation of plasticity-related genes mediate the adaptation of hippocampal synaptic circuits to cope with a stressful environment. In line with this hypothesis, we observed an increase in dendritic length, dendritic complexity and dendritic spine numbers of apical dendrites of CA3 pyramidal neurons in response to ELS, similar to observations in the prefrontal cortex of rats after ELS exposure (Bock et al. 2005a). These experiments as well as pharmacological studies using corticosterone administration or glucocorticosterone receptor agonists indicate that these structural changes are related to the activation of the HPA axis and mediated by stress-induced elevations of corticosterone (Oda and Huttenlocher 1974; Bock et al. 2005a; Monroy et al. 2010; Jafari et al. 2012; Komatsuzaki et al. 2012). Also, it has been reported that corticosterone binding to GR recruits a chromatin remodeling complex in the nucleus and partly contributes to enhanced HAT activity (Schoneveld et al. 2004). Although for our data a direct causality between the histone modifications and neuronal structural changes has yet to be shown, there is evidence from some recent pharmacological studies, which revealed that the inhibition of HDACs induces changes of pre- (synapsin-1, synaptophysin) and post-synaptic (dendritic spines) structures and sprouting of dendrites (Fischer et al. 2007; Calfa et al. 2012; Ricobaraza et al. 2012; Fass et al. 2013) and that spine density is reduced in HDAC2 over-expression mice (Guan et al. 2009).