Unraveling the epigenomic and transcriptomic interplay during alcohol-induced anxiolysis.

Positive effects of alcohol drinking such as anxiolysis and euphoria appear to be a crucial factor in the initiation and maintenance of alcohol use disorder (AUD). However, the mechanisms that lead from chromatin reorganization to transcriptomic changes after acute ethanol exposure remain unknown. Here, we used Assay for Transposase-Accessible Chromatin followed by high throughput sequencing (ATAC-seq) and RNA-seq to investigate epigenomic and transcriptomic changes that underlie anxiolytic effects of acute ethanol using an animal model. Analysis of ATAC-seq data revealed an overall open or permissive chromatin state that was associated with transcriptomic changes in the amygdala after acute ethanol exposure. We identified a candidate gene, Hif3a (Hypoxia-inducible factor 3, alpha subunit), that had 'open' chromatin regions (ATAC-seq peaks), associated with significantly increased active epigenetic histone acetylation marks and decreased DNA methylation at these regions. The mRNA levels of Hif3a were increased by acute ethanol exposure, but decreased in the amygdala during withdrawal after chronic ethanol exposure. Knockdown of Hif3a expression in the central nucleus of amygdala attenuated acute ethanol-induced increases in Hif3a mRNA levels and blocked anxiolysis in rats. These data indicate that chromatin accessibility and transcriptomic signatures in the amygdala after acute ethanol exposure underlie anxiolysis and possibly prime the chromatin for the development of AUD.

Persistence of cerebellar ataxia during chronic ethanol exposure is associated with epigenetic up-regulation of Fmr1 gene expression in rat cerebellum.

BACKGROUND: Alcohol intoxication produces ataxia by affecting the cerebellum, which coordinates movements. Fragile X mental retardation (FMR) protein is a complex regulator of RNA and synaptic plasticity implicated in fragile X-associated tremor/ataxia syndrome, which features ataxia and increased Fmr1 mRNA expression resulting from epigenetic dysregulation of FMRP. We recently demonstrated that acute ethanol-induced ataxia is associated with increased cerebellar Fmr1 gene expression via histone modifications in rats, but it is unknown whether similar behavioral and molecular changes occur following chronic ethanol exposure. Here, we investigated the effects of chronic ethanol exposure on ataxia and epigenetically regulated changes in Fmr1 expression in the cerebellum. METHODS: Male adult Sprague-Dawley rats were trained on the accelerating rotarod and then fed with chronic ethanol or a control Lieber-DeCarli diet while undergoing periodic behavioral testing for ataxia during ethanol exposure and withdrawal. Cerebellar tissues were analyzed for expression of the Fmr1 gene and its targets using a real-time quantitative polymerase chain reaction assay. The epigenetic regulation of Fmr1 was also investigated using a chromatin immunoprecipitation assay. RESULTS: Ataxic behavior measured by the accelerating rotarod behavioral test developed during chronic ethanol treatment and persisted at both the 8-h and 24-h withdrawal time points compared to control diet-fed rats. In addition, chronic ethanol treatment resulted in up-regulated expression of Fmr1 mRNA and increased activating epigenetic marks H3K27 acetylation and H3K4 trimethylation at 2 sites within the Fmr1 promoter. Finally, measurement of the expression of relevant FMRP mRNA targets in the cerebellum showed that chronic ethanol up-regulated cAMP response element binding (CREB) Creb1, Psd95, Grm5, and Grin2b mRNA expression without altering Grin2a, Eaa1, or histone acetyltransferases CREB binding protein (Cbp) or p300 mRNA transcripts. CONCLUSIONS: These results suggest that epigenetic regulation of Fmr1 and subsequent FMRP regulation of target mRNA transcripts constitute neuroadaptations in the cerebellum that may underlie the persistence of ataxic behavior during chronic ethanol exposure and withdrawal.

Essential role for neuronal nitric oxide synthase in acute ethanol-induced motor impairment.

The cerebellum is widely known as a motor structure because it regulates and controls motor learning, coordination, and balance. However, it is also critical for non-motor functions such as cognitive processing, sensory discrimination, addictive behaviors and mental disorders. The cerebellum has the highest relative abundance of neuronal nitric oxide synthase (nNos) and is sensitive to ethanol. Although it has been demonstrated that the interaction of γ-aminobutyric acid (GABA) and nitric oxide (NO) might play an important role in the regulation of ethanol-induced cerebellar ataxia, the molecular mechanisms through which ethanol regulates nNos function to elicit this behavioral effect have not been studied extensively. Here, we investigated the dose-dependent effects of acute ethanol treatment on motor impairment using the rotarod behavioral paradigm and the alterations of nNos mRNA expression in cerebellum, frontal cortex (FC), hippocampus and striatum. We also examined the link between acute ethanol-induced motor impairment and nNos by pharmacological manipulation of nNos function. We found that acute ethanol induced a dose-dependent elevation of ethanol blood levels which was associated with the impairment of motor coordination performance and decreased expression of cerebellar nNos. In contrast, acute ethanol increased nNos expression in FC but did not to change the expression for this enzyme in striatum and hippocampus. The effects of acute ethanol were attenuated by l-arginine, a precursor for NO and potentiated by 7-nitroindazole (7-NI), a selective inhibitor of nNos. Our data suggests that differential regulation of nNos mRNA expression in cerebellum and frontal cortex might be involved in acute ethanol-induced motor impairment.

Essential Role of Histone Methyltransferase G9a in Rapid Tolerance to the Anxiolytic Effects of Ethanol.

BACKGROUND: Tolerance to ethanol-induced anxiolysis promotes alcohol intake, thus contributing to alcohol use disorder development. Recent studies implicate histone deacetylase-mediated histone H3K9 deacetylation in regulating neuropeptide Y expression during rapid ethanol tolerance to the anxiolytic effects of ethanol. Furthermore, the histone methyltransferase, G9a, and G9a-mediated H3K9 dimethylation (H3K9me2) have recently emerged as regulators of addiction and anxiety; however, their role in rapid ethanol tolerance is unknown. Therefore, we investigated the role of G9a-mediated H3K9me2 in neuropeptide Y expression during rapid ethanol tolerance. METHODS: Adult male rats were administered one injection of n-saline followed by single acute ethanol injection (1 g/kg) 24 hours later (ethanol group) or 2 injections (24 hours apart) of either n-saline (saline group) or ethanol (tolerance group). Anxiety-like behaviors and global and Npy-specific G9a and H3K9me2 levels in the amygdala were measured. Effects of G9a inhibitor (UNC0642) treatment on behavioral and epigenetic measures were also examined. RESULTS: Acute ethanol produced anxiolysis and decreased global H3K9me2 and G9a protein levels in the central and medial nucleus of the amygdala as well as decreased occupancy levels of H3K9me2 and G9a near a putative binding site for cAMP-response element binding protein on the Npy gene. Two identical doses of ethanol produced no behavioral or epigenetic changes relative to controls, indicating development of rapid ethanol tolerance. Interestingly, treatment with UNC0642, before the second ethanol dose reversed rapid ethanol tolerance, decreased global H3K9me2 and increased neuropeptide Y levels in the central and medial nucleus of the amygdala. CONCLUSIONS: These results implicate amygdaloid G9a-mediated H3K9me2 mechanisms in regulating rapid tolerance to the anxiolytic effects of ethanol via neuropeptide Y expression regulation.

Effect of Histone Deacetylase Inhibitor on Ethanol Withdrawal-Induced Hyperalgesia in Rats.

BACKGROUND: Increased pain sensitivity is observed following alcohol withdrawal, and attempts to alleviate this hyperalgesia can contribute to the cycle of addiction. The aim of this study was to determine if alcohol withdrawal-induced hyperalgesia was observed in a chronic ethanol exposure model and if this pain was affected by histone deacetylase inhibitors, thus revealing an epigenetic mechanism. METHODS: Adult male Sprague Dawley rats received Lieber-DeCarli liquid control or ethanol (9% v/v) diet for 15 days. Mechanical sensitivity was measured with von Frey hair stimulation of the hindpaw during ethanol administration and 24- and 72-hour withdrawal. RESULTS: Ethanol withdrawal produced severe and sustained mechanical hyperalgesia, an effect not observed in the control or ethanol-maintained groups. Furthermore, this hyperalgesia was attenuated by the histone deacetylase inhibitor, suberoylanilide hydroxamic acid treatment. CONCLUSIONS: Heightened pain sensitivity was observed following withdrawal from chronic ethanol exposure, and histone deacetylase inhibitors could be novel treatments for this alcohol withdrawal-induced hyperalgesia.

Histone Deacetylase Inhibitor Suberanilohydroxamic Acid Treatment Reverses Hyposensitivity to γ-Aminobutyric Acid in the Ventral Tegmental Area During Ethanol Withdrawal.

BACKGROUND: The ventral tegmental area (VTA) is important for alcohol-related reward and reinforcement. Mouse VTA neurons are hyposensitive to γ-aminobutyric acid (GABA) during ethanol (EtOH) withdrawal, and GABA responsiveness is normalized by in vitro treatment with histone deacetylase inhibitors (HDACi). The present study examined the effect of a systemically administered HDACi, suberanilohydroxamic acid (SAHA) on GABA sensitivity, and related molecular changes in VTA neurons during withdrawal after chronic EtOH intake in rats. METHODS: Sprague Dawley male adult rats were fed with Lieber-DeCarli diet (9% EtOH or control diet) for 16 days. Experimental groups included control diet-fed and EtOH diet-fed (0- or 24-hour withdrawal) rats treated with either SAHA or vehicle injection. Single-unit recordings were used to measure the response of VTA neurons to GABA. Immunohistochemistry was performed to examine levels of HDAC2, acetylated histone H3 lysine 9 (acH3K9), and GABAA receptor α1 and α5 subunits in the VTA; quantitative polymerase chain reaction was performed to examine the mRNA levels of HDAC2 and GABAA receptor subunits. RESULTS: VTA neurons from the withdrawal group exhibited GABA hyposensitivity. In vivo SAHA treatment 2 hours before sacrifice normalized the sensitivity of VTA neurons to GABA. EtOH withdrawal was associated with increased HDAC2 and decreased acH3K9 protein levels; SAHA treatment normalized acH3K9 levels. Interestingly, no significant change was observed in the mRNA levels of HDAC2. The mRNA levels, but not protein levels, of GABAA receptor α1 and α5 subunits were increased during withdrawal. CONCLUSIONS: Withdrawal from chronic EtOH exposure results in a decrease in GABA-mediated inhibition, and this GABA hyposensitivity is normalized by in vivo SAHA treatment. Disruption of signaling in the VTA produced by alteration of GABA neurotransmission could be 1 neuroadaptive physiological process leading to craving and relapse. These results suggest that HDACi pharmacotherapy with agents like SAHA might be an effective treatment for alcoholism.

Adolescent Alcohol Exposure-Induced Changes in Alpha-Melanocyte Stimulating Hormone and Neuropeptide Y Pathways via Histone Acetylation in the Brain During Adulthood.

Background: Adolescent intermittent ethanol exposure causes long-lasting alterations in brain epigenetic mechanisms. Melanocortin and neuropeptide Y signaling interact and are affected by ethanol exposure in the brain. Here, the persistent effects of adolescent intermittent ethanol on alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and their regulation by histone acetylation mechanisms were investigated in adulthood. Methods: Male rats were exposed to adolescent intermittent ethanol (2 g/kg, i.p.) or volume-matched adolescent intermittent saline from postnatal days 28 to 41 and allowed to grow to postnatal day 92. Anxiety-like behaviors were measured by the elevated plus-maze test. Brain regions from adult rats were used to examine changes in alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and the histone acetylation status of their promoters. Results: Adolescent intermittent ethanol-exposed adult rats displayed anxiety-like behaviors and showed increased pro-opiomelanocortin mRNA levels in the hypothalamus and increased melanocortin 4 receptor mRNA levels in both the amygdala and hypothalamus compared with adolescent intermittent saline-exposed adult rats. The alpha-Melanocyte stimulating hormone and melanocortin 4 receptor protein levels were increased in the central and medial nucleus of the amygdala, paraventricular nucleus, and arcuate nucleus of the hypothalamus in adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Neuropeptide Y protein levels were decreased in the central and medial nucleus of the amygdala of adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Histone H3K9/14 acetylation was decreased in the neuropeptide Y promoter in the amygdala but increased in the melanocortin 4 receptor gene promoter in the amygdala and the melanocortin 4 receptor and pro-opiomelanocortin promoters in the hypothalamus of adolescent intermittent ethanol-exposed adult rats compared with controls. Conclusions: Increased melanocortin and decreased neuropeptide Y activity due to changes in histone acetylation in emotional brain circuitry may play a role in adolescent intermittent ethanol-induced anxiety phenotypes in adulthood.

Chronic Alcohol Exposure Differentially Alters One-Carbon Metabolism in Rat Liver and Brain.

BACKGROUND: Epigenetic mechanisms such as DNA methylation play an important role in regulating the pathophysiology of alcoholism. Chronic alcohol exposure leads to behavioral changes as well as decreased expression of genes associated with synaptic plasticity. In the liver, it has been documented that chronic alcohol exposure impairs methionine synthase (Ms) activity leading to a decrease in S-adenosyl methionine/S-adenosyl homocysteine (SAM/SAH) ratio which results in DNA hypomethylation; however, it is not known whether similar alterations of SAM and SAH levels are also produced in brain. METHODS: Male adult Sprague Dawley rats were fed chronically with Lieber-DeCarli ethanol (EtOH) (9% v/v) or control diet. The EtOH-diet-fed rats were withdrawn for 0 and 24 hours. The cerebellum and liver tissues were dissected and used to investigate changes in one-carbon metabolism, SAM, and SAH levels. RESULTS: We found that chronic EtOH exposure decreased SAM levels, SAM/SAH ratio, Ms, methylene tetrahydrofolate reductase, and betaine homocysteine methyltransferase (Bhmt) expression and increased methionine adenosyltransferase-2b (Mat2b) but not Mat2a expression in the liver. In contrast, chronic EtOH exposure decreased SAH levels, increased SAM/SAH ratio and the expression of Mat2a and S-adenosyl homocysteine hydrolase, while the levels of SAM or Bhmt expression in cerebellum remained unaltered. However, in both liver and cerebellum, chronic EtOH exposure decreased the expression of Ms and increased Mat2b expression. All chronic EtOH-induced changes of one-carbon metabolism in cerebellum, but not liver, returned to near-normal levels during EtOH withdrawal. CONCLUSIONS: These results indicate a decreased "methylation index" in liver and an increased "methylation index" in cerebellum. The opposing changes of the "methylation index" suggest altered DNA methylation in liver and cerebellum, thus implicating one-carbon metabolism in the pathophysiology of alcoholism.

Adolescent alcohol exposure alters lysine demethylase 1 (LSD1) expression and histone methylation in the amygdala during adulthood.

Alcohol exposure in adolescence is an important risk factor for the development of alcoholism in adulthood. Epigenetic processes are implicated in the persistence of adolescent alcohol exposure-related changes, specifically in the amygdala. We investigated the role of histone methylation mechanisms in the persistent effects of adolescent intermittent ethanol (AIE) exposure in adulthood. Adolescent rats were exposed to 2 g/kg ethanol (2 days on/off) or intermittent n-saline (AIS) during postnatal days (PND) 28-41 and used for behavioral and epigenetic studies. We found that AIE exposure caused a long-lasting decrease in mRNA and protein levels of lysine demethylase 1(Lsd1) and mRNA levels of Lsd1 + 8a (a neuron-specific splice variant) in specific amygdaloid structures compared with AIS-exposed rats when measured at adulthood. Interestingly, AIE increased histone H3 lysine 9 dimethylation (H3K9me2) levels in the central nucleus of the amygdala (CeA) and medial nucleus of the amygdala (MeA) in adulthood without producing any change in H3K4me2 protein levels. Acute ethanol challenge (2 g/kg) in adulthood attenuated anxiety-like behaviors and the decrease in Lsd1 + 8a mRNA levels in the amygdala induced by AIE. AIE caused an increase in H3K9me2 occupancy at the brain-derived neurotrophic factor exon IV promoter in the amygdala that returned to baseline after acute ethanol challenge in adulthood. These results indicate that AIE specifically modulates epizymes involved in H3K9 dimethylation in the amygdala in adulthood, which are possibly responsible for AIE-induced chromatin remodeling and adult psychopathology such as anxiety.

Role of Growth Arrest and DNA Damage-Inducible, Beta in Alcohol-Drinking Behaviors.

BACKGROUND: The contribution of epigenetic factors, such as histone acetylation and DNA methylation, to the regulation of alcohol-drinking behavior has been increasingly recognized over the last several years. GADD45b is a protein demonstrated to be involved in DNA demethylation at neurotrophic factor gene promoters, including at brain-derived neurotrophic factor (Bdnf) which has been highly implicated in alcohol-drinking behavior. METHODS: DNA methyltransferase-1 (Dnmt1), 3a, and 3b, and Gadd45a, b, and g mRNA were measured in the nucleus accumbens (NAc) and ventral tegmental areas of high ethanol (EtOH) consuming C57BL/6J (C57) and low alcohol consuming DBA/2J (DBA) mice using quantitative reverse transcriptase polymerase chain reaction (PCR). In the NAc, GADD45b protein was measured via immunohistochemistry and Bdnf9a mRNA using in situ PCR. Bdnf9a promoter histone H3 acetylated at lysines 9 and 14 (H3K9,K14ac) was measured using chromatin immunoprecipitation, and 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) using methylated DNA immunoprecipitation. Alcohol-drinking behavior was evaluated in Gadd45b haplodeficient (+/-) and null mice (-/-) utilizing drinking-in-the-dark (DID) and 2-bottle free-choice paradigms. RESULTS: C57 mice had lower levels of Gadd45b and g mRNA and GADD45b protein in the NAc relative to the DBA strain. C57 mice had lower NAc shell Bdnf9a mRNA levels, Bdnf9a promoter H3K9,K14ac, and higher Bdnf9a promoter 5HMC and 5MC. Acute EtOH increased GADD45b protein, Bdnf9a mRNA, and histone acetylation and decreased 5HMC in C57 mice. Gadd45b +/- mice displayed higher drinking behavior relative to wild-type littermates in both DID and 2-bottle free-choice paradigms. CONCLUSIONS: These data indicate the importance of the DNA demethylation pathway and its interactions with histone posttranslational modifications in alcohol-drinking behavior. Further, we suggest that lower DNA demethylation protein GADD45b levels may affect Bdnf expression possibly leading to altered alcohol-drinking behavior.

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

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.

Reversal of deficits in dendritic spines, BDNF and Arc expression in the amygdala during alcohol dependence by HDAC inhibitor treatment.

Development of anxiety-like behaviours during ethanol withdrawal has been correlated with increased histone deacetylase (HDAC) activity and decreased brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated protein (Arc) gene expression in the amygdala. Furthermore, HDAC-mediated histone modifications play a role in synaptic plasticity. In this study we used the HDAC inhibitor trichostatin A (TSA) to determine whether HDAC inhibition could prevent ethanol withdrawal-induced deficits in dendritic spine density (DSD), BDNF or Arc expression in the amygdala of rats. It was found that decreased BDNF and Arc expression in the central (CeA) and medial nucleus of amygdala (MeA), observed during withdrawal after chronic ethanol exposure, were normalized following acute TSA treatment. TSA treatment was also able to attenuate anxiety-like behaviours during ethanol withdrawal and correct the observed decrease in DSD in the CeA and MeA of ethanol-withdrawn rats. Taken together, these findings demonstrate that correcting the deficits in histone acetylation through TSA treatment also amends downstream synaptic plasticity-related deficits such as BDNF and Arc expression, and DSD in the CeA and MeA as well as attenuates anxiety-like behaviours in rats during withdrawal after chronic ethanol exposure.

Effects of histone deacetylase inhibitors on amygdaloid histone acetylation and neuropeptide Y expression: a role in anxiety-like and alcohol-drinking behaviours.

Recent studies have demonstrated the involvement of epigenetic mechanisms in psychiatric disorders, including alcoholism. Here, we investigated the effects of histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) on amygdaloid HDAC-induced histone deacetylation and neuropeptide Y (NPY) expression and on anxiety-like and alcohol-drinking behaviours in alcohol-preferring (P) and -non-preferring (NP) rats. It was found that P rats displayed higher anxiety-like and alcohol-drinking behaviours, higher amygdaloid nuclear, but not cytosolic, HDAC activity, which was associated with increased HDAC2 protein levels and deficits in histone acetylation and NPY expression in the central (CeA) and medial nucleus of amygdala (MeA), as compared to NP rats. TSA treatment attenuated the anxiety-like and alcohol-drinking behaviours, with concomitant reductions in amygdaloid nuclear, but not cytosolic HDAC activity, and HDAC2, but not HDAC4, protein levels in the CeA and MeA of P rats, without effect in NP rats. TSA treatment also increased global histone acetylation (H3-K9 and H4-K8) and NPY expression in the CeA and MeA of P, but not in NP rats. Histone H3 acetylation within the NPY promoter was also innately lower in the amygdala of P rats compared with NP rats; which was normalized by TSA treatment. Voluntary ethanol intake in P, but not NP rats, produced anxiolytic effects and decreased the HDAC2 levels and increased histone acetylation in the CeA and MeA. These results suggest that higher HDAC2 expression-related deficits in histone acetylation may be involved in lower NPY expression in the amygdala of P rats, and operative in controlling anxiety-like and alcohol-drinking behaviours.

Effects of acute ethanol exposure on anxiety measures and epigenetic modifiers in the extended amygdala of adolescent rats.

Epigenetic mechanisms appear to play an important role in neurodevelopment. We investigated the effects of acute ethanol exposure on anxiety measures and function of histone deacetylases (HDAC) and DNA methyltransferases (DNMT) in the amygdala and bed nucleus of stria terminalis (BNST) of adolescent rats. One hour after ethanol exposure, rats were subjected to anxiety measures. A subset of adolescent rats was exposed to two doses (24 h apart) of ethanol (2 g/kg) to measure rapid ethanol tolerance to anxiolysis. The HDAC and DNMT activities and mRNA levels of DNMT isoforms were measured in the amygdala and BNST. The lower dose of ethanol (1 g/kg) produced neither anxiolysis, nor inhibited the HDAC and DNMT activities in the amygdala and BNST, except DNMT activity in BNST was attenuated. Anxiolysis by ethanol was observed at 2 and 2.25 g/kg, whereas higher doses (2.5 and 3 g/kg) were found to be sedative. DNMT activity in the amygdala and BNST, and nuclear HDAC activity in the amygdala, but not in the BNST were also inhibited by these doses of ethanol. A lack of tolerance was observed on ethanol-induced inhibition of DNMT activity in the amygdala and BNST, and nuclear HDAC activity in the amygdala, as well to anxiolysis produced by ethanol (2 g/kg). The DNMT1, DNMT3a, and DNMT3b mRNA expression in the amygdala was not affected by either 1or 2 doses of 2 g/kg. However, DNMT1 and DNMT3a expression in the BNST was increased, whereas DNMT3l mRNA was decreased in the amygdala, after 2 doses of 2 g/kg ethanol. These results suggest that reduced sensitivity to anxiolysis and the lack of rapid tolerance to the anxiolytic effects of ethanol and inhibition of HDAC and DNMT functions may play a role in engaging adolescents in binge drinking patterns.

EZH2-dependent epigenetic reprogramming in the central nucleus of amygdala regulates adult anxiety in both sexes after adolescent alcohol exposure.

Alcohol use and anxiety disorders occur in both males and females, but despite sharing similar presentation and classical symptoms, the prevalence of alcohol use disorder (AUD) is lower in females. While anxiety is a symptom and comorbidity shared by both sexes, the common underlying mechanism that leads to AUD and the subsequent development of anxiety is still understudied. Using a rodent model of adolescent intermittent ethanol (AIE) exposure in both sexes, we investigated the epigenetic mechanism mediated by enhancer of zeste 2 (EZH2), a histone methyltransferase, in regulating both the expression of activity-regulated cytoskeleton-associated protein (Arc) and an anxiety-like phenotype in adulthood. Here, we report that EZH2 protein levels were significantly higher in PKC-δ positive GABAergic neurons in the central nucleus of amygdala (CeA) of adult male and female rats after AIE. Reducing protein and mRNA levels of EZH2 using siRNA infusion in the CeA prevented AIE-induced anxiety-like behavior, increased H3K27me3, decreased H3K27ac at the Arc synaptic activity response element (SARE) site, and restored deficits in Arc mRNA and protein expression in both male and female adult rats. Our data indicate that an EZH2-mediated epigenetic mechanism in the CeA plays an important role in regulating anxiety-like behavior and Arc expression after AIE in both male and female rats in adulthood. This study suggests that EZH2 may serve as a tractable drug target for the treatment of adult psychopathology after adolescent alcohol exposure.

Sunitinib induces cardiotoxicity through modulating oxidative stress and Nrf2-dependent ferroptosis in vitro and in vivo.

SUN, a multi-targeted tyrosine kinase inhibitor, exerts cardiotoxicity which hinders its clinical use. It is necessary to elucidate molecular mechanism of SUN-induced cardiotoxicity. To elucidate molecular mechanism of SUN-induced cardiotoxicity and whether it is related to Nrf2-dependent ferroptosis, in vitro model with H9c2 cells derived from rat heart tissue and in vivo model (C57BL/6J male mouse) were used in the present study. In vivo model was established by oral treatment of SUN at dose of 10, 20, 40 mg/kg for 14 days. Body weight, ECG, plasma enzyme activities, histology staining were performed to evaluate heart function. Western-blot was performed to analyze the level of ferroptosis-related proteins. In vitro results indicated that SUN markedly induced ferroptosis embodied as collapsed MMP, accumulated iron and elevated ROS. In vivo results showed that SUN significantly impaired cardiac function. Abnormal electrocardiogram, increased serum CK and lactate LDH levels were significantly observed in SUN groups. Histology staining showed that SUN caused structural injuries and fibrosis deposition. Moreover, SUN increased the level of MDA and Fe2+ content, decreased the level of GSH. Both in vitro and in vivo experiments indicated that SUN reduced the expression of Nrf2, HO-1, NQO1, GPX4 and FTH1, enhanced the TfR expression. This study suggested that oxidative stress and Nrf2-dependent ferroptosis played a vital role in SUN-induced cardiotoxicity.

Transcriptional Dysregulation of Cholesterol Synthesis Underlies Hyposensitivity to GABA in the Ventral Tegmental Area During Acute Alcohol Withdrawal.

BACKGROUND: The ventral tegmental area (VTA) is a dopaminergic brain area that is critical in the development and maintenance of addiction. During withdrawal from chronic ethanol exposure, the response of VTA neurons to GABA (gamma-aminobutyric acid) is reduced through an epigenetically regulated mechanism. In the current study, a whole-genome transcriptomic approach was used to investigate the underlying molecular mechanism of GABA hyposensitivity in the VTA during withdrawal after chronic ethanol exposure. METHODS: We performed RNA sequencing of the VTA of Sprague Dawley male rats withdrawn for 24 hours from a chronic ethanol diet as well as sequencing of the VTA of control rats fed the Lieber-DeCarli diet. RNA sequencing data were analyzed using weighted gene coexpression network analysis to identify modules that contained coexpressed genes. Validation was performed with quantitative polymerase chain reaction, gas chromatography-mass spectrometry, and electrophysiological assays. RESULTS: Pathway and network analysis of weighted gene coexpression network analysis module 1 revealed a significant downregulation of genes associated with the cholesterol synthesis pathway. Consistent with this association, VTA cholesterol levels were significantly decreased during withdrawal. Chromatin immunoprecipitation indicated a decrease in levels of acetylated H3K27 at the transcriptional control regions of these genes. Electrophysiological studies in VTA slices demonstrated that GABA hyposensitivity during withdrawal was normalized by addition of exogenous cholesterol. In addition, inhibition of cholesterol synthesis produced GABA hyposensitivity, which was reversed by adding exogenous cholesterol to VTA slices. CONCLUSIONS: These results suggest that decreased expression of cholesterol synthesis genes may regulate GABA hyposensitivity of VTA neurons during alcohol withdrawal. Increasing cholesterol levels in the brain may be a novel avenue for therapeutic intervention to reverse detrimental effects of chronic alcohol exposure.

Histone deacetylases (HDAC)-induced histone modifications in the amygdala: a role in rapid tolerance to the anxiolytic effects of ethanol.

BACKGROUND: Rapid tolerance to the anxiolytic effects of ethanol appears to be an important factor in the development of alcoholism. Here, we investigated the involvement of amygdaloid histone deacetylases (HDAC)-induced epigenetic changes in rapid ethanol tolerance (RET). METHODS: RET in rats was induced by 2 ethanol injections administered 24 hours apart. Both ethanol-tolerant and control rats were treated with the HDAC inhibitor, trichostatin A (TSA), and anxiety-like behaviors were measured. HDAC activity, histone (H3 and H4) acetylation, and neuropeptide Y (NPY) expression in the amygdala of these rats were also measured. RESULTS: A single ethanol exposure was able to produce an anxiolytic response, inhibit amygdaloid HDAC activity, and increase both histone acetylation and NPY expression (mRNA and protein levels) in the central nucleus of amygdala (CeA) and medial nucleus of amygdala (MeA) of rats. In contrast, 2 exposures of the same dose of ethanol (24 hours apart) neither elicited a similar anxiolytic response nor modulated HDAC activity, histone acetylation, or NPY expression in the amygdala. However, exposure to a higher dose of ethanol on the second day was able to produce an anxiolytic response and also inhibit amygdaloid HDAC activity. TSA treatment caused the reversal of RET by inhibiting HDAC activity, thereby increasing histone acetylation and NPY expression in the CeA and MeA. CONCLUSIONS: Cellular tolerance to the initial acute ethanol-induced inhibition of HDAC activity and the subsequent upregulation of histone acetylation and NPY expression in the amygdala may be involved in the mechanisms underlying rapid tolerance to the anxiolytic effects of ethanol.

Acute and Protracted Prenatal Stress Produce Mood Disorder-Like and Ethanol Drinking Behaviors in Male and Female Adult Offspring.

Background: Alcohol use disorder (AUD) is a complex and chronic relapsing brain disease, which is often co-morbid with psychiatric disorders such as anxiety and depression. AUD phenotypes differ in men and women. Although genetic factors play an important role in its pathophysiology, epidemiologic evidence suggests that during prenatal development, individuals are more vulnerable to the negative effects of environmental factors that may predispose them to AUD later in life. We explored the effects of prenatal stress on the development of AUD phenotypes as well as anxiety- and depression-like behaviors using rat model. Methods: In this study, timed-pregnant Sprague Dawley dams were used. Dams in the control group were left undisturbed throughout gestation, whereas dams in stress groups were either subjected to protracted or acute restraint stress under bright light. At adulthood, the anxiety-like, ethanol drinking, and sucrose drinking behaviors were measured using the Light/Dark Box test and two-bottle free-choice procedure. Results: Compared to the control group, both the male and female offspring in the stress groups exhibited anxiety-like behavior and consumed significantly higher amounts of ethanol in which the acute stress group demonstrated the higher ethanol preference. Moreover, male but not female offspring from the stress groups had decreased sucrose preferences. Conclusion: These findings suggest that protracted and acute prenatal stress in late pregnancy can induce in anxiety-, depressive-like behaviors, and excessive ethanol intake in adult offspring.

Targeted epigenomic editing ameliorates adult anxiety and excessive drinking after adolescent alcohol exposure.

Adolescent binge drinking is a major risk factor for psychiatric disorders later in life including alcohol use disorder. Adolescent alcohol exposure induces epigenetic reprogramming at the enhancer region of the activity-regulated cytoskeleton-associated protein (Arc) immediate-early gene, known as synaptic activity response element (SARE), and decreases Arc expression in the amygdala of both rodents and humans. The causal role of amygdalar epigenomic regulation at Arc SARE in adult anxiety and drinking after adolescent alcohol exposure is unknown. Here, we show that dCas9-P300 increases histone acetylation at the Arc SARE and normalizes deficits in Arc expression, leading to attenuation of adult anxiety and excessive alcohol drinking in a rat model of adolescent alcohol exposure. Conversely, dCas9-KRAB increases repressive histone methylation at the Arc SARE, decreases Arc expression, and produces anxiety and alcohol drinking in control rats. These results demonstrate that epigenomic editing in the amygdala can ameliorate adult psychopathology after adolescent alcohol exposure.