Elsevier

Neurobiology of Disease

Volume 82, October 2015, Pages 607-619
Neurobiology of Disease

Potential role of adolescent alcohol exposure-induced amygdaloid histone modifications in anxiety and alcohol intake during adulthood

https://doi.org/10.1016/j.nbd.2015.03.019Get rights and content

Highlights

  • Adolescent intermittent ethanol (AIE) decreased amygdaloid histone H3 acetylation.

  • AIE produced long-lasting upregulation of HDAC2 levels and HDAC activity.

  • AIE lowered BDNF and Arc gene expression in adulthood due to histone modifications.

  • HDAC inhibitors lowered AIE-induced anxiety and alcohol intake in adulthood.

  • AIE-induced chromatin remodeling may lead to anxiety and alcoholism later in life.

Abstract

Binge drinking is common during adolescence and can lead to the development of psychiatric disorders, including alcoholism in adulthood. Here, the role and persistent effects of histone modifications during adolescent intermittent ethanol (AIE) exposure in the development of anxiety and alcoholism in adulthood were investigated. Rats received intermittent ethanol exposure during post-natal days 28–41, and anxiety-like behaviors were measured after 1 and 24 h of the last AIE. The effects of AIE on anxiety-like and alcohol-drinking behaviors in adulthood were measured with or without treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Amygdaloid brain regions were collected to measure HDAC activity, global and gene-specific histone H3 acetylation, expression of brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated (Arc) protein and dendritic spine density (DSD). Adolescent rats displayed anxiety-like behaviors after 24 h, but not 1 h, of last AIE with a concomitant increase in nuclear and cytosolic amygdaloid HDAC activity and HDAC2 and HDAC4 levels leading to deficits in histone (H3-K9) acetylation in the central (CeA) and medial (MeA), but not in basolateral nucleus of amygdala (BLA). Interestingly, some of AIE-induced epigenetic changes such as, increased nuclear HDAC activity, HDAC2 expression, and decreased global histone acetylation persisted in adulthood. In addition, AIE decreased BDNF exons I and IV and Arc promoter specific histone H3 acetylation that was associated with decreased BDNF, Arc expression and DSD in the CeA and MeA during adulthood. AIE also induced anxiety-like behaviors and enhanced ethanol intake in adulthood, which was attenuated by TSA treatment via normalization of deficits in histone H3 acetylation of BDNF and Arc genes. These novel results indicate that AIE induces long-lasting effects on histone modifications and deficits in synaptic events in the amygdala, which are associated with anxiety-like and alcohol drinking behaviors in adulthood.

Introduction

Binge drinking is prevalent during adolescence (Donovan, 2004, Matthews, 2010, O'Malley et al., 1998, Wechsler et al., 2000). Both clinical and preclinical studies have shown that alcohol use during adolescence leads to a greater risk for developing alcoholism and other psychiatric disorders in adulthood (DeWit et al., 2000, Guerri and Pascual, 2010, Grant and Dawson, 1997). Adolescence is an important developmental period during which the brain undergoes maturation including changes in neurotransmission, gene expression, and synaptic remodeling, specifically, the formation and pruning of axons, dendrites, and synapses in various brain regions (Craiu, 2013, Fumagalli et al., 2007, Tau and Peterson, 2010, Spear, 2013). In general, the central (CeA), medial (MeA), and basolateral (BLA) nuclei of the amygdala have been shown to be involved in the processes that regulate emotion, anxiety, and alcoholism (Koob and Volkow, 2010, LeDoux, 2000, Pandey et al., 2006, Whalen et al., 2001). One major hypothesis of alcohol dependence involves alterations in the allostatic state driven by negative emotional adaptations within the circuitry of the amygdala that is characterized as the “dark side of addiction” (Koob, 2013, Koob, 2003, Koob and Volkow, 2010).

Epigenetic processes, such as histone acetylation and DNA methylation mechanisms, have been shown to play a role in neuromaturation by contributing to the stability of gene expression during brain development (Mehler, 2008, MacDonald and Roskams, 2008, Murgatroyd and Spengler, 2011, Szyf, 2013, Witt, 2010). Well-studied epigenetic mechanisms include histone modifications via lysine acetylation where histone deacetylases (HDACs) remove acetyl groups from histones, leading to a condensed chromatin state, blocking transcriptional activator access to DNA and thereby decreasing gene transcription (de Ruijter et al., 2003, Feng and Fan, 2009, Gräff and Tsai, 2013, Jenuwein and Allis, 2001, Thiagalingam et al., 2003). Several genes, such as brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated (Arc) protein are involved in regulating dendritic spine density (DSD) in the brain and are epigenetically controlled by histone acetylation (Bramham and Messaoudi, 2005, Maddox et al., 2013, Moonat et al., 2013, Tsankova et al., 2004). We recently reported that higher expression of HDAC2, but not HDAC1, 3, 4, or 5 isoforms regulates deficits in histone H3-K9 acetylation, BDNF and Arc expression and DSD in the CeA and MeA of alcohol preferring (P) compared with alcohol non-preferring (NP) adult rats, and HDAC2 in the CeA is mechanistically involved in anxiety-like and alcohol-drinking behaviors of P rats (Moonat et al., 2013). Additionally, withdrawal after chronic ethanol exposure was associated with reduced histone (H3&H4) acetylation, reduced expression of BDNF and Arc and decreased DSD in the CeA and MeA as well as upregulation of HDAC activity in the amygdala of adult Sprague–Dawley (SD) rats (Pandey et al., 2008a, You et al., 2014). Blocking up-regulation of HDAC activity with trichostatin A (TSA) treatment during ethanol withdrawal attenuated anxiety-like behaviors and restored deficits in histone acetylation (Pandey et al., 2008a). These results suggest that amygdaloid HDAC-induced chromatin remodeling may be involved in the process of alcohol preference and dependence in adult rats.

It has been shown that epigenetic modifications in the developing brain, such as histone modifications and DNA methylation due to exposure to environmental stimuli or early life adversity, may lead to both short- and long-term changes in synaptic plasticity that affect behaviors in adulthood (Fagiolini et al., 2009, Morris et al., 2010, Roth et al., 2009, Szyf et al., 2008). Currently, it is unknown how epigenetic factors interact with early life alcohol exposure to shape emotional brain circuitries during development and their role in anxiety and alcoholism in adulthood. We therefore investigated effects of adolescent intermittent ethanol (AIE) treatment on HDAC-mediated histone deacetylation in the amygdala and anxiety-like behaviors in adolescent rats, and if AIE-induced histone modifications can persist in adulthood and coordinate shifts in synaptic plasticity-associated gene expression in the amygdala and regulate anxiety-like and alcohol-drinking behaviors. Furthermore, we investigated the effects of TSA on the reversal of AIE-induced behavioral phenotypes and changes in gene-specific histone H3 acetylation in the amygdala during adulthood.

Section snippets

Animals and intermittent ethanol exposure

Pregnant female Sprague–Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN, USA) and maintained in 12:12 h light/dark cycle with ad libitum access to water and food. All animal experimental procedures followed the NIH guidelines for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee. Male pups were weaned at the post-natal day (PND) 21 from dams and were group-housed (2 or 3 rats) in cages at the same animal facility

Effect of AIE on anxiety-like behaviors during adolescence

To examine the effects of AIE on the anxiety-like behaviors, rats were subjected to LDB and EPM exploration tests after 1 h and 24 h from the last AIE or AIS (Figs. 1A, B). One-way ANOVA indicated significant group differences for time spent in light and dark compartments of LDB (F2, 32 = 7.1, p < 0.01). Although AIE adolescent rats did not display anxiety-like behaviors at 1 h (ethanol on-board) as compared to AIS rats, anxiety-like behaviors were observed after 24 h of the last AIE injection (AIW).

Discussion

The findings of the present study indicate that AIE produced anxiety-like behaviors during withdrawal that persist and are associated with alcohol-drinking behaviors in adulthood. We provided novel evidence that AIE produced HDAC-induced histone H3 deacetylation due to higher HDAC activity and increased protein levels of HDAC2 and 4 in the amygdala during adolescence. Some of these changes, such as increased nuclear HDAC activity and HDAC2 levels and decreased histone H3-K9 acetylation

Conclusions

The data presented here indicate that AIE produced deficits in histone H3-K9 acetylation due to increased expression of HDAC2 and 4 isoforms and increased HDAC activity in the amygdala, which is correlated with anxiety-like behaviors during adolescence. Interestingly, anxiety-like behaviors induced by AIE persist in adulthood and are associated with an increase in alcohol intake. These AIE-induced behavioral phenotypes are correlated with deficits in global and gene specific (BDNF and Arc)

Conflict of interest

SCP reports that a US patent application entitled “Histone acetyl transferase activators and histone deacetylase inhibitors in the treatment of alcoholism” (serial number 60/848237 filed on September 29th, 2006) is currently pending. Other authors reported no biomedical financial interests or potential conflicts of interest.

Acknowledgments

This work was supported by the National Institute on Alcohol Abuse and Alcoholism Grants AA-019971 (NADIA project), AA-010005, and AA-013341, and by the Department of Veterans Affairs [Merit Review Grant (I01BX000143); Research Career Scientist award] to S.C.P.

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