Paternal alcohol exposure reduces alcohol drinking and increases behavioral sensitivity to alcohol selectively in male offspring

Abstract

Alcohol use disorder (AUD) is heritable, but the genetic basis for this disease remains poorly understood. Although numerous gene variants have been associated with AUD, these variants account for only a small fraction of the total risk. The idea of inheritance of acquired characteristics, i.e. "epigenetic inheritance," is re-emerging as a proven adjunct to traditional modes of genetic inheritance. We hypothesized that alcohol drinking and neurobiological sensitivity to alcohol are influenced by ancestral alcohol exposure. To test this hypothesis, we exposed male mice to chronic vapor ethanol or control conditions, mated them to ethanol-naïve females, and tested adult offspring for ethanol drinking, ethanol-induced behaviors, gene expression, and DNA methylation. We found that ethanol-sired male offspring had reduced ethanol preference and consumption, enhanced sensitivity to the anxiolytic and motor-enhancing effects of ethanol, and increased Bdnf expression in the ventral tegmental area (VTA) compared to control-sired male offspring. There were no differences among ethanol- and control-sired female offspring on these assays. Ethanol exposure also decreased DNA methylation at the BdnfÆpromoter of sire's germ cells and hypomethylation was maintained in the VTA of both male and female ethanol-sired offspring. Our findings show that paternal alcohol exposure is a previously unrecognized regulator of alcohol drinking and behavioral sensitivity to alcohol in male, but not female, offspring. Paternal alcohol exposure also induces epigenetic alterations (DNA hypomethylation) and gene expression changes that persist in the VTA of offspring. These results provide new insight into the inheritance and development of alcohol drinking behaviors.

Figure 1. Chronic vapor ethanol exposure in sires.

(A) 8-week-old C57BL/6J mice were exposed to EtOH vapor or room air for 8 hours/day, 5 days/week, for 5 weeks and immediately housed with 2 EtOH-naïve Strain 129Sv/ImJ females for 48 hours; after mating, they were re-exposed for 3 days and motile sperm was collected. (B) Blood EtOH concentrations showed limited variability across 5 weeks and averaged 147.1+/−7.52 mg/dl (mean ± SEM) (n = 25). (C) There was no difference in weight gain between EtOH (n = 25) and Room air (n = 27) exposed sires during the 5 weeks of exposure. Data presented as mean ± SEM bars.

Figure 2. Comparison of E-sired and C-sired litters.

(A) There were no differences in number of litters, number of male and female offspring, or (B) number of offspring per litter between E- and C-sires. (C) E-sired male offspring (n = 40) gained significantly more weight after weaning at 3 weeks and maintained increased weight through week 6 compared to C-sired male offspring (n = 61) (p<0.001). (D) There was no significant difference in weight between E- (n = 43) and C-sired (n = 45) female offspring. Data presented as mean ± SEM (Note: Error bars in C and D are obscured by the data points), **p<0.01, ***p<0.001.

Figure 3. Offspring were tested for EtOH consumption vs. water on a 2 bottle, free choice drinking assay.

(A) E-sired male offspring (n = 17) had significantly decreased preference for EtOH compared to C-sired male offspring (n = 17) as well as (B) decreased EtOH consumption and (C) no change in total volume consumption per body weight. There were no significant differences between E-sired female offspring (n = 12) and C-sired female offspring (n = 12) on (D) EtOH preference, (E) EtOH consumption, or (F) total volume consumed. Data presented as mean ± SEM, *p<0.05, **p<0.01.

Figure 4. Offspring were tested for EtOH-induced anxiolysis by comparing performance on an elevated plus maze 10 minutes after i.p. injection of 1 g/kg EtOH or saline.

(A) E-sired male offspring spend greater time in open arms after treatment with EtOH compared to C-sired male offspring and E-sired male offspring treated with saline; E-sired male offspring treated with EtOH also have (B) increased percent of open arm entries and (C) total arm entries relative to C-sired male offspring and E-sired male offspring treated with saline. There were no significant differences between E- and C-sired females treated with EtOH or saline on (D) time spent in open arms, (E) percent open arm entries, or (F) total arm entries. n = 6–7/group, data presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Offspring were tested for performance on an accelerating rotarod 35 minutes after i.p. injection of 1/kg EtOH or saline.

(A) E-sired male offspring treated with EtOH performed significantly better on the 5^th trial compared to those treated with saline. There are no significant differences associated with EtOH treatment in (B) C-sired male offspring, (C) E-sired female offspring, or (D) C-sired female offspring. n = 7–8/group, data presented as mean ± SEM, *p<0.05.

Figure 6. Expression of Bdnf and Dlk1 were measured in the VTA of offspring and DNA methylation was measured at the Bdnf exon IXa promoter in motile sperm and offspring VTA.

(A) E-sired male offspring had significantly increased expression of Bdnf exon IXa but not Dlk1 or Bdnf exon IV in the VTA relative to C-sired males. (B) There were no significant differences in Bdnf or Dlk1 expression between E- and C-sired females in the VTA. (C) EtOH-exposed sires had decreased DNA methylation at the Bdnf exon IXa promoter relative to Room air exposed sires. (D) Male and (E) female E-sired offspring had decreased DNA methylation at the Bdnf exon IXa promoter relative to C-sired offspring. N.D., none detected, n = 4–7/group, data presented as mean ± SEM, *p<0.05, ***p<0.001.