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Keywords:

  • Arc;
  • chromatin remodeling;
  • early life stress;
  • Egr1;
  • epigenetic

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

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.

Abbreviations used
Arc

activity-regulated cytoskeleton-association protein

Egr1

early growth response protein

ELS

early life stress

GR

glucocorticoid receptor

HAT

histone acetyltransferase

HPA

hypothalamic–pituitary–adrenal axis

LTP

long-term potentiation

MS

maternal separation

N-ChIP

native chromatin immunoprecipitation

PFC

prefrontal cortex

PND

post-natal day

qPCR

quantitative polymerase chain reaction

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).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Animals and maternal separation

C57BL/6 mice were used in all experiments and housed on a 12-h light-dark cycle with access to food and water ad libitum. After birth of the pups, the home cages were not cleaned during the first 16 post-natal days to avoid stress for the dams and pups. The newborn mice were culled to 6 or 8 per litter (male/female was 3/3 or 4/4) and housed together with their mother. We applied repeated maternal separation during post-natal day (PND) 14–16 as early life stressor, since this paradigm results in increased spine density and dendritic length (Bock et al. 2005a), changes of mRNA for steroid receptor subtypes and altered long-term potentiation (LTP) (Wang et al. 2012). The day of birth was defined as PND 0 and whole litters were randomly assigned to the following experimental groups:

Control group

Pups of this group were reared undisturbed together with their mother and siblings until the time of the respective experiment.

Early life stress group (MS)

From PND 14 to 16, pups were separated from their mother and isolated individually in an 8-cube holding box (13 cm × 13 cm for each cube, covered with soft paper bedding) for 3 h (10:00 AM–13:00 PM). All animals were handled in accordance with the European Communities Council Directive of November 1986 (86/609/EEC).

mRNA expression

Male pups were killed at two different time points: (i) 30 min after the end of the last separation period, or (ii) 120 min after the end of the last separation period. The hippocampus was immediately frozen in liquid nitrogen. RNA extraction was performed using RNeasy plus mini kit (QIAGEN GmbH, Hilden, Germany). Genomic DNA was eliminated using on column DNase treatment and RNA samples were reverse transcribed into cDNA using high capacity cDNA reverse transcription kit including Rnase inhibitor (Applied Biosystems, Darmstadt, Germany). Quantitative PCR (q-PCR) was performed on a StepOnePlus Real-time PCR System (Applied Biosystems) using TaqMan gene expression assays, including Arc (Mm00479619_g1, Applied Biosystems), Egr1 (Mm00656724_m1, Applied Biosystems) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) (Mm01545399_m1, Applied Biosystems). Arc and Egr1 mRNA levels were calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001), and Hprt1 was selected as internal control. The final value was expressed as relative value to the control group.

Immunoblotting

Thirty minutes after the end of the last separation period, the hippocampus was dissected and frozen immediately. A protocol for nuclear extracts (Abcam protocol with small modifications) was applied. The tissue was homogenized followed by centrifugation and the sediment was re-suspended in 1 mL of Triton extraction buffer (TEB, 1x PBS containing 0.5% Triton × 100, 2 mM phenylmethylsulfonyl fluoride, 0.02% NaN3). Then the lysate was placed on ice for 10 min followed by centrifugation at 6500 g for 10 min at 4C to spin down the nuclei. To extract nuclear proteins the pellet was re-suspended in 0.2 N HCl and incubated overnight at 4C. The supernatant was collected after 10 min of centrifugation at 6500 g at 4°C. Protein concentration was measured by using BCA protein assay kit (Novagen®; Merck, Darmstadt, Germany).

Ten micrograms of the lysates was subjected to a 4–15% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (Bio-Rad Laboratories, München, Germany) and then blotted to a Polyvinylidene diflouride mebrane (PVDM) (0.2 micron, Roth, Karlsruhe, Germany) at 30V for 2 h. The blots were blocked in Roti block (Roth) working solution followed by incubation with primary antibodies (anti-acetyl-histone H3, 1 : 10 000, Millipore, Billirica, USA; anti-acetyl-histone H4, 1 : 4000, Millipore; anti-nucleolin, 1 : 1000, Abcam, Cambridge, UK) for overnight at 4°C. The blots were incubated in horseradish peroxidase conjugated anti-rabbit antibody (1 : 4000, 12–348, Millipore). After covering with Luminata crescendo western horseradish peroxidase substrate (Millipore) the blot images were visualized using a Fuji dark room with CCD camera system (FUJIFILM, LAS-1000, Fujifilm Europe, Düsseldorf, Germany). Optical density was measured using ImageJ 1.41o National Institutes of Health, USA. To control the sample loading, optical density was normalized to the nuclear control protein nucleolin.

Native chromatin immunoprecipitation quantitative PCR (N-ChIP-qPCR)

Thirty minutes after the end of the last separation period, the hippocampus was frozen immediately in liquid nitrogen. We applied a ChIP protocol as described before (Matevossian and Akbarian 2008) with some modifications. The frozen tissue was ground in liquid nitrogen, homogenized and treated with micrococcal nuclease (MNase) (USB) reaction buffer. The reaction was terminated by adding 0.5 M EDTA. After hypotonization and pre-cleansing with protein A sepharose beads, lysates were split into groups of input (500 μL), no antibody control (1500 μL), and the antibody (1500 μL). Antibody (anti-acetyl histone H3, 06-599; anti-acetyl histone H4, 06-866; Millipore) and no antibody groups were incubated at 4°C overnight. Antibody-specific complexes were precipitated by adding protein A sepharose beads for 1 h at 4°C. Specific antibody bound chromatin fragments were eluted from beads using 0.1 M NaHCO3 containing 1% SDS. Then, the proteins were digested at 65°C overnight, DNA was extracted with phenol–chloroform and purified with MinElute reaction cleanup kit (Qiagen).

Subsequent qPCR was performed using TaqMan assays (Applied Biosystems) using primers specific for the promoter regions of Arc or Egr1 (TIB, for primer sequences, see Guan et al. 2009). Beta-globin (5′-TGACCAATAGCCTCA GAGTCCTG-3′ and 5′-GAAGACCTGTCCTTTTATTCTTCACC-3′, 6FAM-ACCTT GCTCCCCTTACCCC-TCC-BBQ) was used as locus control region (inactivated control gene). Each PCR reaction was run in triplicates for each sample. The immunoprecipitation data were normalized to input as an index of transcription.

Corticosterone measurement

To confirm that the maternal separation (MS) procedure elevates stress hormones, blood levels of serum corticosterone were measured. Blood was collected in Serum-Gel Z tube (SARSTEDT, Nümbrecht, Germany) immediately after decapitation. Corticosterone levels were measured using corticosterone rat/mouse ELISA kit (DEV9922, Demeditec, Kiel-Wellsee, Germany).

Quantitative analysis of hippocampal neuronal morphology

Male mice were decapitated on PND 21, the brains rapidly removed and unfixed brains were immersed in Golgi-Cox solution for 14 days. Brain sections of 150 μm were processed and developed as described in previous studies (i.e., Bock et al. 2005a). For each animal (eight animals per experimental group), three neurons of the following neuron types were analyzed; (i) Pyramidal neurons located in the hippocampal CA3 subregion (ii) granular neurons in the dentate gyrus. All neurons were reconstructed using a computer-based neuron tracing system (NEUROLUCIDA®, MicroBright-Field, Williston, VT, USA) and the following parameters were quantified: (i) proximal, medial, and terminal dendritic length; (ii) proximal, medial, and terminal spine number; (iii) dendritic complexity. Proximal, medial, and terminal segments were defined by applying a Sholl analysis (Sholl 1953) with concentric rings, 50 μm distance apart from each other. All measurements were performed by an experimenter, who was unaware of the experimental condition of the animals/materials.

Statistics

For gene expression two-way anova was used with time point and treatment as parameters. For western blot, ChIP-qPCR data and histological data two-tailed t-test was applied. All data were presented as means ± SEM. Significance was set at  0.05 for all data sets.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Increased serum corticosterone levels in stressed animals

Immediately after the last separation session, the MS pups showed a significant 2.5-fold increase in the serum corticosterone level compared to the non-stressed pups from the control group (Fig. 1, = 0.004).

image

Figure 1. Elevated serum corticosterone in response to early life stress (ELS) (maternal separation, MS). Corticosterone levels measured directly after the last stress exposure are significantly increased compared to controls (= 7 for controls and = 11 for MS). (* 0.05).

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Increased Arc mRNA and Egr1 mRNA expression in stressed animals

Arc mRNA levels in the hippocampus were measured at different time points 30 and 120 min after the last session of MS. Two-way anova revealed an effect for treatment, reflected by a significant increase in Arc and Egr1 mRNA expression in stressed animals compared to unstressed controls (Arc: F(1,44) = 8.928, = 0.005; Egr1: F(1,44) = 7.297, = 0.01). No effects for the factor time point (Arc: F(1,44)=1.588, = 0.214; Egr1: F(1,44)=0.328, = 0.570), and no treatment × time point interaction (Arc: F(1,44)=1.588, = 0.214; Egr1: F(1,44)=0.328, = 0.570) were found.

Increased acetylation of histones H3 and H4 in stressed animals

Quantitative western blot analysis was applied to test whether ELS induces rapid, immediate alterations in the acetylation levels of H3 and H4. Our results show that 30 min after the end of the last exposure to MS, there was a significant increase both in the levels of acetylated H3 and the levels of acetylated H4 in the hippocampus of stressed animals (Fig. 2b, = 0.05 and = 0.013, respectively).

image

Figure 2. Histone acetylation-induced elevation of Arc and Egr1 mRNA expression in the hippocampus immediately after early life stress (ELS) (maternal separation, MS). (a) Gene Expression. Arc and Egr1 mRNA expression is increased in the MS group compared to the unstressed control group (n = 12 per group). (b) Histone Acetylation. Both acetylated histone H3 and H4 are elevated in the MS group (H3Ac: n = 10 per group; H4Ac: n = 11 per group). (c) Histone H3 acetylation associated with Egr1 and Arc expression. No changes of histone H3 acetylation at the promoter regions of Arc and Egr1 after MS (n = 10 per group). (d) Histone H4 acetylation associated with Egr1 and Arc expression. MS significantly increases histone H4 acetylation at the promoter regions of Arc and Egr1 (n = 11 per group). (* 0.05).

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Increased acetylation of histone H4 but not H3 at the promotor region of Arc and Egr1 in stressed animals

To test the hypothesis that the enhanced Arc and Egr1 mRNA expression was regulated by increased acetylation of H3 and H4, we applied N-ChIP-qPCR to quantify the associations of acetylated H3 and H4 to the promoter regions of Arc and Egr1. Overall, we found that the increased expression of Arc and Egr1 was associated with enhanced acetylation of histone H4, which was evident at the promoter region of Egr1 and Arc in the MS group (Fig. 2d, < 0.05). In contrast, acetylation of H3 was not significantly altered at the promoter regions of Arc and Egr1 (Fig. 2c).

Increased dendritic complexity and spine number in hippocampal CA3 neurons of stressed animals

A significant increase in dendritic complexity was found in MS mice compared to controls, indicated by a higher number of intersections in apical dendrites of CA3 pyramidal neurons (Fig. 3c, = 0.029). Similarly, a significant increase of CA3 proximal dendritic length was observed in MS mice (Fig. 3d, = 0.04), while medial and terminal apical dendritic length showed trend toward increase (= 0.07 and = 0.08, respectively). While there was no change in spine density, the total number of dendritic spines was significantly increased on the medial and terminal dendritic segments (Fig. 3e, = 0.02 and = 0.007. No dendritic and synaptic changes were found in granular neurons of the dentate gyrus (data not shown).

image

Figure 3. Increased dendritic length, dendritic complexity, and dendritic spine number of CA3 apical dendrites after early life stress (ELS). (a) Photographic image of Golgi-Cox stained pyramidal CA3 neurons. (b) Representative examples of three-dimensionally reconstructed apical dendrites of CA3 pyramidal neuron in the control and MS group. (c) MS induces significant increases in the complexity of CA3 apical dendrites. (d) MS induces significant increases in CA3 proximal apical dendritic segments. (e) MS induces a significant increase in the number of spines on medial and terminal apical dendritic segments. (n = 8 per group, * 0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

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).

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

In conclusion, we demonstrate for the first time that early life stress up-regulates the histone acetylation levels around the promoter regions of the synaptic plasticity genes Arc and Egr1, resulting in an increased expression of these genes. It is tempting to speculate that these epigenetic mechanisms interfere with the establishment and ‘formatting’ of hippocampal circuits and thereby induce long-lasting structural changes in hippocampal synaptic connectivity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

We thank Susann Becker for expert technical assistance with the histological material. This study was supported by grants from the Bundesministerium für Bildung und Forschung (BMBF, TUR10/I48 to KB and 01KR1207D to JB), and a grant from the federal state of Saxony-Anhalt and the “European Regional Development Fund” (ERDF2007-2013), Vorhaben: Center for Behavioral Brain Sciences (CBBS) FKZ:1211080005. The authors declare no conflict of interest.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References
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