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. 2017 Sep 26:10:307.
doi: 10.3389/fnmol.2017.00307. eCollection 2017.

Intermittent Ethanol during Adolescence Leads to Lasting Behavioral Changes in Adulthood and Alters Gene Expression and Histone Methylation in the PFC

Jennifer T Wolstenholme  1   2 , Tariq Mahmood  1 , Guy M Harris  1 , Shahroze Abbas  1 , Michael F Miles  1   2   3
Affiliations

Intermittent Ethanol during Adolescence Leads to Lasting Behavioral Changes in Adulthood and Alters Gene Expression and Histone Methylation in the PFC

Jennifer T Wolstenholme et al. Front Mol Neurosci. .

Abstract

Adolescents primarily consume alcohol in binges, which can be particularly harmful to the developing frontal cortex and increase risk for an adult alcohol use disorder. We conducted a study investigating immediate and long lasting changes to the prefrontal cortex (PFC) transcriptome to determine the molecular mechanisms underlying adult ethanol behavioral sensitivity following binge ethanol in adolescence. DBA/2J mice were orally dosed with 4 g/kg ethanol intermittently from day 29 to 42. Adolescent mice were tested for anxiety-like behavior and ethanol sensitivity using the loss of righting reflex task. As adults, mice were tested for cognitive changes using the novel object recognition task, ethanol-induced anxiolysis and ethanol sensitivity. Adolescent binge ethanol altered ethanol sensitivity in young mice and led to lasting memory deficits in the object recognition test and greater ethanol sensitivity in adulthood. Using genomic profiling of transcripts in the PFC, we found that binge ethanol reduced myelin-related gene expression and altered chromatin modifying genes involved in histone demethylation at H3K9 and H3K36. We hypothesize that ethanol's actions on histone methylation may be a switch for future transcriptional changes that underlie the behavioral changes lasting into adulthood.

Keywords: adolescent; epigenetics; ethanol; genomics; prefrontal cortex.

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Figures

FIGURE 1
FIGURE 1
Binge ethanol in adolescence increases locomotor activity in adolescence. Ethanol treatment did not alter the percent time in the light (A) or the percent distance traveled in the light (B) in the light–dark box 24 h after the last ethanol dose at PND 43 (n = 7–10/group). Time spent ambulatory (C) and total locomotor distance traveled (D) in the 5-min test were significantly increased in ethanol exposed males and females. Data is presented as mean +/– SEM. ∗p < 0.05, main effect of treatment by Two-Way ANOVA.
FIGURE 2
FIGURE 2
Adolescent ethanol reduces ethanol sensitivity in adolescent DBA/2J mice. (A) DBA/2J males (n = 9/group) and females (n = 6–7/group) take a similar amount of time to lose their righting reflex after high doses of ethanol. (B) Both binge treated males and females showed a shorter LORR duration after a high dose of ethanol at PND 46. Data is presented as mean +/– SEM. ∗p < 0.05, main effect of treatment by Two-Way ANOVA.
FIGURE 3
FIGURE 3
Recognition memory was impaired in adult mice after binge ethanol in adolescence. After a 5-min delay between training and testing (A), ethanol-exposed DBA/2J adult mice (n = 6–11/group) had a zero or negative discrimination index, indicating a failure to recognize a novel object, or failure to remember a familiar object. After a 1-h delay (B), DBA/2J adult female mice had a trend to a lower discrimination index. Data is presented as mean +/– SEM. ∗p < 0.05, main effect of treatment by Two-Way ANOVA. ˆp = 0.07, a trend for a significant effect of treatment.
FIGURE 4
FIGURE 4
Binge ethanol does not alter ethanol-induced anxiolysis in adulthood. Ethanol (2 g/kg i.p.) significantly increased time spent in the light (A) and distance traveled in the light (B) as compared to adult saline treated mice (n = 5–10/group). Sex differences or prior exposure to ethanol did not modify anxiety-like behavior in the light–dark box. (C) Total distance traveled during the task was higher in ethanol treated mice. Females previously exposed to ethanol during adolescence had significantly higher locomotor activity as compared to all other groups. Data is presented as mean +/– SEM. ∗p < 0.05, main effect of ethanol injection by Three-Way ANOVA. #p < 0.05 for interaction between adolescent exposure, ethanol injection and sex by Three-Way ANOVA.
FIGURE 5
FIGURE 5
Ethanol sensitivity is greater in ethanol-exposed mice in adulthood. (A) Latency to lose the righting reflex was significantly shorter in adult females (n = 13/group) as compared to males (n = 19–20/group). ∗p < 0.05, main effect of sex by Two-Way ANOVA. But did not differ between treatment groups. (B) In adulthood, adolescent ethanol exposure increased LORR duration as adults. Data is presented as mean +/– SEM. ∗p < 0.05, main effect of treatment by Two-Way ANOVA.
FIGURE 6
FIGURE 6
Binge ethanol during adolescents alters mRNA expression of myelin-related genes. (A) Binge ethanol decreased many myelin-related genes including Mag, Mbp, Mobp and Plp mRNA in male and female DBA/2J PFC by qPCR (n = 4–6, ∗p < 0.05, interaction between treatment and sex by Two-Way ANOVA). In adolescence, DBA/2J males have more myelin-related mRNA than females (#p < 0.05, main effect of sex by Two-Way ANOVA). (B) In adulthood, prior binge ethanol did not alter myelin-related gene expression. Sex differences were not found in adults. Data is presented as mean +/– SEM.
FIGURE 7
FIGURE 7
H3K36 methylation is decreased in ethanol exposed adolescent males. (A) Using ELISAs to measure global methylation in PFC (n = 3–4/group), binge ethanol decreased H3K36me, H3K36me2 and H3K36me3 in DBA males (∗p < 0.05, interaction between treatment and sex by Two-Way ANOVA). (B) H3K9 methylation (mono- di- or tri-) was not significantly altered by binge ethanol (n = 3–4/group). Data is presented as mean +/– SEM.
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