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Addict Biol. Author manuscript; available in PMC 2023 January 01.
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Published in final edited form as:

Addict Biol. 2022 January ; 27(1): e13078. doi:10.1111/adb.13078.

Paternal alcohol exposure reduces acquisition of operant

alcohol self-administration and affects Bdnf DNA methylation in
male and female offspring
Steven J. Nieto, PhD1, Colin N. Haile, MD, PhD1, Cana B. Quave, BA1, Mark J. Harding, BA2,
David A. Nielsen, PhD2, Richard A. Meisch, MD, PhD1, Therese A. Kosten, PhD1
1University of Houston, Department of Psychology & Texas Institute for Measurement, Evaluation
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and Statistics (TIMES)

2Baylor College of Medicine, The Menninger Department of Psychiatry and Behavioral Sciences

Abstract
Familial transmission of alcohol use disorder reflects genetic and environmental factors. Paternal
alcohol exposure may affect rodent offspring via epigenetic modifications transmitted through the
male germ line. While such exposure alters alcohol sensitivity in mouse offspring, no studies
examined if it impacts the development of operant alcohol self-administration in rats. We exposed
male (sires) Wistar rats to chronic intermittent ethanol in vapor chambers (16 h/day; 5 days/week)
or to air for 6 weeks. Eight weeks later, rats were mated with alcohol-naive females. Adult
alcohol- and control-sired F1 offspring were assessed in acquisition of alcohol self-administration
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in which increasing alcohol concentrations (2.5%, 5%, & 10%, v/v) were delivered after one
lever press (fixed ratio 1 or FR1). Prior to alcohol sessions, rats were trained to lever press for
food delivery under an FR1 schedule of reinforcement. DNA methylation levels of the brain
derived neurotrophic factor (Bdnf) gene were measured in sperm, nucleus accumbens (NAc),
and medial prefrontal cortex (mPFC) in sires and in offspring. Alcohol-exposed sires had lower
Bdnf DNA methylation levels in NAc and greater methylation levels in mPFC. Although this
pattern was not recapitulated in offspring, alcohol-sired offspring of both sexes did show aberrant
Bdnf DNA methylation patterns compared to control-sired offspring. Alcohol-sired offspring self-
administered less alcohol (5% & 10%) with no group differences in food responding. Results
indicate that paternal alcohol exposure prior to conception protects against alcohol’s initial
reinforcing effects but the pattern of dysregulated Bdnf methylation in reward-related circuitry
did not mimic changes seen in sires.
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Corresponding author: Dr. Therese A. Kosten, Professor, Dept. of Psychology & TIMES, Health and Biomedical Sciences Building
1, 4849 Calhoun Road Room 432, Houston, TX 77204-6022, Office: 713-743-3724, takosten@central.uh.edu.
Author contributions
SJN designed and ran the experiments, analyzed and interpreted the data, and wrote the manuscript. CNH and CBQ assisted in
collecting behavioral data and brain tissue and drafting the manuscript. DAN and MJH ran the DNA methylation experiments and
made contributions to writing the manuscript. RAM made substantial contributions to the study design and provided important
intellectual comments to the manuscript. TAK made substantial contributions to the design of the project, data interpretation, and
writing the manuscript.
Conflict of Interest
There are no actual or potential conflicts of interest.
 Nieto et al. Page 2

Keywords
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sex differences; epigenetics; operant conditioning; reinforcement; preconception; rats

1. Introduction
Parental alcohol misuse has intergenerational consequences, including altered alcohol
sensitivity in offspring. Children of parents with Alcohol Use Disorder (AUD) have lower
sensitivity to motor-impairing and stress-activating effects of alcohol 1–5 and a higher risk
of AUD 6. While maternal alcohol misuse is well-studied, the impact of paternal alcohol
drinking is less understood 7–9. However, paternal (sire) alcohol exposure sex-dependently
alters sensitivity to alcohol in rodent offspring. Male, but not female, offspring of alcohol-
sired (A-sired) mice exhibit greater anxiolysis to alcohol 10. A-sired male mice offspring
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also show lower alcohol preference and consumption in two-bottle procedures 10 and exhibit
conditioned preference to a low dose and conditioned aversion to a moderate dose of alcohol
11. These studies corroborate research that show male rat offspring of cocaine-exposed sires

exhibit reduced cocaine-seeking 12. To our knowledge, no studies utilizing rats have assessed
the initial sensitivity to the reinforcing effects of alcohol in A-sired offspring using operant
techniques.

That altered sensitivity to alcohol is seen mainly in male offspring suggests heritable risks
pass through the male germ line 10. Although genetic variants that contribute to AUD
are not fully delineated, genome-wide association studies identify several single-nucleotide
polymorphisms associated with AUD 13. Technical and replication limitations however
question the significance of these findings 14. Further, genetic variants identified thus far
only explain ~0.1% of the heritable risk for AUD 15. The missing heritability needs to be
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explained by alternative factors.

Epigenetic processes alter gene expression without changing the DNA sequence and likely
contribute to heritability of AUDs potentially through environmentally-induced epigenetic
modifications. These mechanisms are reversible and can recapitulate in successive mitotic
generation of cells 16. DNA methylation is a well-studied mechanism known to increase or
decrease gene transcription depending upon the genomic location. Promoter regions of genes
contain a high density of CpG dinucleotides 17. Addition of methyl groups to cytosines near
transcription start sites can decrease gene transcription and prevent binding of transcription
factors 18. In rodents, the length of spermatogenesis is less than 60 days 19, which allows
for epigenetic changes induced by the paternal environment to be reflected in male germ
cells. Alcohol alters DNA methylation levels of imprinted and non-imprinted loci in sperm
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that can pass to offspring 10,20–22. For example, alcohol lowers methylation of brain derived
neurotrophic factor (Bdnf) gene in sire sperm that is maintained in the ventral tegmental area
(VTA) of offspring 10. Although offspring of both sexes inherit lower Bdnf methylation
levels, only A-sired males exhibit decreased alcohol consumption. These findings are
consistent with previous work showing that BDNF is a protective neurotrophic factor that
dampens alcohol-dependent phenotypes and keeps alcohol drinking in moderation 23–25.
While paternal alcohol-induced epigenetic changes may influence alcohol consumption in

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offspring, whether it affects sensitivity to the reinforcing effects of alcohol when alcohol
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availability is response-contingent is not known.

Operant self-administration procedures provide valuable information beyond what is

assessed with two-bottle and place conditioning procedures; both appetitive and
consummatory behaviors can be measured 26–28. The acquisition phase of drug and alcohol
self-administration provides information related to initial stages of the addiction process
that is difficult to investigate in humans 29. Acquisition procedures have identified genetic,
epigenetic, and environmental factors that affect vulnerability or resilience to drug use 29–34
leading to a better understanding of the etiology of AUD.

The purpose of this study was to determine if paternal alcohol exposure alters acquisition
of operant alcohol self-administration in rat offspring. Based on previous findings, we
hypothesized that A-sired male, but not female, offspring will show delayed acquisition of
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alcohol self-administration. This hypothesis was tested with a procedure modeled after our
prior work assessing acquisition of cocaine self-administration in rats with neonatal isolation
experience 31. A second purpose was to identify if alcohol-exposed sires differed in Bdnf
DNA methylation levels in sperm, nucleus accumbens (NAc), and medial prefrontal cortex
(mPFC) and determine if these changes were maintained in sperm and brains of offspring.
We postulated that Bdnf methylation levels will be lower in sperm and NAc and greater in
the mPFC, brain regions involved in reward-related processes, in alcohol-exposed sires and
that this epigenetic profile would be maintained in sperm and brains of offspring.

2. Materials and Methods

2.1. Animals and housing
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Male and female Wistar rats (Charles River, Wilmington, MA) were used to generate
offspring. Sires (400–500 g) were pair-housed prior to mating and sacrificed shortly after
dams were confirmed pregnant. Dams were group-housed prior to mating and then sacrificed
after offspring were weaned. Offspring were group-housed (females) or pair-housed (males)
after weaning and throughout the course of the study. Most animals were housed in amber
polysulfone cages and kept in a temperature and humidity-controlled vivarium. During
chronic intermittent ethanol vapor exposure, male sires were placed in standard rat cages
housed within vapor chambers. The vivarium was maintained on a 12:12 light/dark cycle
(lights on at 7:00 AM). Animals had ad libitum access to food and water except during
operant procedures as noted below. The University of Houston Institutional Animal Care and
Use Committee approved the experimental protocol in accordance with guidelines set forth
in the “Guide for the Care and Use of Laboratory Animals 8th Edition”.
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Adult male rats (sires) were exposed to alcohol vapor (n = 10) or room air (n = 10) prior to
being mated with alcohol naïve females (n = 20). A-sired (n = 9 males; n = 10 females) and
C-sired (n = 10 males; n = 8 females) offspring were trained to lever press for sucrose pellets
and then alcohol solution as described below. A separate cohort of A-sired (n = 10 males;
n = 10 females) and C-sired offspring (n = 9 males; n = 10 females) were used for DNA
methylation studies.

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2.2. Solution and drug preparations

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Alcohol (ethyl alcohol, 190 proof, USP grade, Koptec, King of Prussia, PA) was mixed with
tap water to reach concentrations of 2.5% (v/v), 5% (v/v), and 10% (v/v) alcohol.

2.3. Paternal chronic intermittent ethanol exposure

Male rats (sires) were made dependent by chronic, intermittent exposure to vapor alcohol
as previously described 35,36. This model reliably induces alcohol dependence as indicated
by negative emotional-like state and somatic symptoms 37. Standard rat cages were housed
inside sealed, transparent plastic chambers into which 95% alcohol vapor was pumped.
Males underwent cycles of 16 h (6pm) on and 8 h (10am) off for five consecutive days per
week over six weeks. Nondependent rats were housed in similar conditions but exposed to
room air. Blood samples were collected from the lateral saphenous vein and blood alcohol
levels (BAL) measured using the AM1 Alcohol Analyser (Analox Instruments, UK). Air
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flow rates were adjusted to achieve the desired BAL range (150–225 mg/dl).

2.4. Breeding and offspring rearing

Male sires were left undisturbed for 8 weeks after their last alcohol vapor or air session.
Thereafter, they were housed with alcohol-naïve females. Females were examined daily
for the presence of a mating plug and, if found, males were removed from the cages and
sperm and brain regions extracted the following day. Litters were culled to 10 pups (5
pups/sex) and weighed at postnatal days [PD] 1, 4, 7, 10, 35, and weekly into adulthood.
Self-administration sessions began when offspring reached adulthood ~PD 75. To control
for possible litter effects, no more than two pups per sex per litter were used in the self-
administration study and one pup per sex per litter was used in the DNA methylation study
(see Figure 1 for experimental timeline).
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2.5. Tissue collection and DNA extraction

Motile sperm was collected from sires and male offspring using the double swim up assay
38. Briefly, the cauda epididymis was dissected from the testes and placed in 1% bovine
serum albumin. Longitudinal cuts were made along the cauda epididymis and it was placed
with 1% bovine serum albumin in a 15 mL conical tube. The tissue was incubated at 37°C
for 30-min. Supernatant containing sperm was collected and incubated again at 37°C for
10-min. The top 1 mL of supernatant was collected and pelleted at 4°C at 4000 RPM for
5-min. Motile sperm was resuspended in sperm lysis buffer with Proteinase K and incubated
overnight at 50°C. DNA was extracted from motile sperm using a modified guanidine
thiocyanate method 39. DNA samples were immediately placed on dry ice and stored at
−80°C until bisulfite treatment.
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Brain tissue (mPFC and NAc) was collected from sires and offspring after rats were
anesthetized with isoflurane and decapitated. Brain regions were dissected using a Rodent
Brain Matrix (RBM-4000C) and immediately placed on dry ice and stored at −80°C. DNA
was extracted using the Gentra Puregene DNA isolation methods (Qiagen, Valencia, CA).

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2.6. DNA methylation

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DNA methylation levels within the Bdnf promoter region were determined using direct
sequencing methods as reported previously 40,41. Genomic DNA (300 ng) was treated with
sodium bisulfite using the EZ-96 DNA Methylation Kit D5004 (Zymo Research, Irvine,
CA) according to the manufacturer’s instructions. Bisulfite-treated DNA was amplified
using a modified step-down method with annealing temperatures of 56, 53, 50, 47, and
44°C with the primers M-RATBDNF-4F (5’-GGTAGAGGAGGTATTATATGATAGT-3’)
and M-RATBDNF-4R (5’-ATAACCCATATATACTCCTATTCTTCAACA-3’). Sequencing
was performed at GENEWIZ, Inc. (South Plainfield, NJ) using both forward and reverse
primers used in the amplification of the Bdnf promoter. Trace files (.ab1) were analyzed
using the Epigenetic Sequencing Methylation Analysis Software (Epigenomics AG; Berlin,
Germany) version 3.2.1. Nucleotides were numbered relative to the exon IV A of the ATG
translation start site. The rat Bdnf gene exon IV promoter region was analyzed for predicted
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transcription factor binding sites using AliBaba2.1 (http://www.generegulation.com/pub/

programs/alibaba2/index.html).

2.7. Self-administration apparatus

Self-administration sessions were conducted in operant chambers placed within sound-
attenuating cubicles equipped with fans (Coulbourn Instruments, Allentown PA). Each
chamber had a house light on one side of the cage and two retractable levers on the
opposite side. Above each lever was a triple cue light and in between the levers were two
access areas. One area was a recessed food receptacle into which sucrose pellets could be
dispensed from a pellet dispenser. A dipper could be immersed in a solution reservoir and
activated to present 0.1 ml of solution through the second recessed area. Both access areas
were equipped with a light and infrared sensors used to detect head entries. Experimental
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parameters were programmed and data tabulation was performed using a software package
(Graphic State Notation, Coulbourn) installed on a PC computer.

2.8. Self-administration training

Rats were water-restricted overnight (7pm to 9am) and then trained to drink water from
the dipper for 2-weeks. Levers were retracted and each session began with two dipper
presentations. After these two “primes” and for the rest of the 30-min sessions, any
head entry into the access area triggered a dipper presentation. Dipper presentation times
gradually decreased from 15- to 3-sec, the duration used for the rest of the study.
Water-restriction and dipper training ended after all rats met the criterion of at least 25
dipper presentations over two consecutive days. Then, rats were food-restricted to 85% of
their free-feeding body weight and trained to press a lever for sucrose pellets (BioServ,
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Flemington, NJ) under a fixed ratio 1 (FR1) schedule of reinforcement (30-min sessions).
Sessions started with the house light illuminated and protrusion of levers into the chamber.
A pellet was dispensed only after the active lever was pressed. Inactive lever presses had no
programmed consequences. Operant training for food-maintained responding was continued
until all rats obtained 20 reinforcers in ≤5-min over two consecutive days. Food restriction
continued throughout the remainder of the study.

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Alcohol self-administration sessions were conducted under similar parameters (30-min

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sessions; FR1 schedule), except that weekly ascending concentrations of alcohol (2.5%
−10%) were available for delivery. Rats began training sessions pressing for 2.5% alcohol
during week 1, 5% during week 2, and 10% during weeks 3–5. To decrease the group
differences between alcohol and control sired offspring when responding for 10% alcohol,
specifically within female offspring, a 5% alcohol concentration was the final alcohol
solution used during the remaining weeks of alcohol training (weeks 6–8). Animals
successfully acquired operant alcohol self-administration when they achieved at least 25
active lever presses and responding was consistent (< 20% variability over two consecutive
days). The numbers of active and inactive lever presses, and alcohol deliveries were
tabulated.

2.9. Statistical Analysis

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The number of pups per sex born per litter were compared using Student t-tests. Body
weights were analyzed using a three-way mixed design analysis of variance with Sex and
Sire as the between group factors and Time as the repeated measure factor. Sessions to
acquisition, active lever presses, and alcohol deliveries were also analyzed using a three-way
mixed design analysis of variance with Sex and Sire as the between groups factor and
Session or Week as the repeated measure factor. DNA methylation data were analyzed using
two-way mixed design analysis of variance with CpG sites considered a within subject factor
and Sire and Sex as between group factors. Tukey post hoc tests were used to follow up on
significant interactions. Statistical analyses were performed using SAS software 9.4 (SAS
Institute, Cary, NC) with statistical significance defined as p<0.05. Data are presented as
mean ± SEM.
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3. Results
3.1. Litter characteristics and offspring body weights
A-sired and C-sired groups did not differ on numbers of litters (10 each), pups per litter
(A-sired: 10.5 + 1.8; C-sired: 12.4 + 1.3), or male (A-sired: 46; C-sired: 52) and female
(A-sired: 49; C-sired 43) offspring per litter, p’s> 0.05 (see Table 1). Body weights of
A-sired and C-sired offspring are shown in Table 2. There were significant main effects of
Sire, F(1, 92) = 49.632, p<0.001, Sex, F(1, 92)= 739.87, p<0.001, and Time, F(5, 460)=
6725.0, p<0.001, along with significant interactions of Time X Sire, F(5, 460)= 14.994,
p<0.001 and Time X Sex, F(5, 460)= 527.49, p<0.001. Tukey post hoc comparisons showed
that A-sired offspring (male and female) weighed less than C-sired offspring specifically at
PD35 and PD75, p’s <0.001. Tukey pairwise comparisons also showed that male offspring
weighed more than females specifically at PD35 and PD75, p’s <0.001.
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3.2. Alcohol Self-administration

Sessions to reach acquisition criteria for water, food, and alcohol did not differ by Sex or
Sire (p’s > 0.05; see Table 3). There were no effects of Sex or Sire group on daily dipper
presentations during water training (p’s > 0.05; see Supplemental Figure 1). For weekly
active lever presses during food training, there was no effect of Sire (p > 0.05) but there was
a significant main effect of Sex, F(1, 92)= 9.271, p =0.008, such that female rats had greater

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active lever presses relative to males (data not shown). There was no significant effect of Sex
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or Sire on daily food deliveries during food training (p’s > 0.05; see Supplemental Figure 2).

Active lever presses emitted by male and female offspring are shown in Figure 2. There was
a significant main effect of Day, F(39, 975)= 6.777, p<0.001, and significant interactions
of Day X Sex, F(39, 975)= 2.970, p<0.001, and Day X Sire X Sex, F(39, 975)= 2.254,
p<0.001. To determine the loci of sire differences, we averaged daily active lever presses
into weekly blocks. Tukey post hoc analyses within each of these weekly blocks showed that
A-sired males pressed the active lever less than C-sired males during weeks 2–3 (p’s < 0.05).
A-sired females pressed the active lever less than C-sired females during weeks 4–7 (p’s <
0.05). Sire and Sex had no effect on the numbers of inactive lever presses (data not shown).

Alcohol deliveries are shown in Figure 3. There was a significant main effect of Day,
F(39, 975)= 4.191, p<0.001, and significant interactions of Day X Sire, F(39, 975)= 1.542,
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p=.019, Day X Sex, F(39, 975)= 3.557, p<0.001, Day X Sire X Sex, F(39, 975)= 1.977,
p<0.001. Tukey post hoc analyses within the weekly time blocks revealed that A-sired males
earned fewer alcohol deliveries compared to C-sired males during weeks 2 and 3 (p’s <
0.05). A-sired females earned fewer alcohol deliveries during weeks 4–7 (p’s < 0.05).

3.3. Bdnf DNA Methylation Levels

Bdnf DNA methylation levels in sires are depicted in Figure 4. In the NAc, there were
significant main effects of Treatment, F(1, 17)= 3.011, p<0.01, and CpG site F(11, 154)
= 9.865, p<0.001. Alcohol-exposed sires had lower DNA methylation levels relative to
air-exposed sires (Fig. 4B). DNA methylation levels varied by CpG site, but there was
no interaction with treatment (p>0.05). In the mPFC, there were significant main effects
of Treatment, F(1, 17)= 3.592, p<0.05, and CpG site (11, 154) = 9.806, p<0.001. Alcohol-
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exposed sires had greater Bdnf DNA methylation levels compared to control sires (Fig. 4D).
DNA methylation levels varied by CpG site, but there was no interaction with treatment
(p=0.126). In sire sperm, there was a significant main effect of CpG site, F(11, 154) = 17.44,
p<0.001, but not Treatment (p>0.05; see Supplemental Figure 3).

Bdnf DNA methylation levels in male and female offspring are depicted in Figure 5. In
the NAc, there was a significant main effect of CpG site, F(11, 264)= 24.465, p<0.05,
and a CpG site X Sire X Sex interaction, F(11, 264) = 2.481, p<0.01. Tukey post-hoc
tests showed that A-sired male offspring had lower methylation at CpG sites −11 and
62 (p<0.05) and higher methylation levels at CpG site 43 (p<0.01; Fig. 5A). A-sired
female offspring also showed differential methylation patterns that varied by CpG sites.
Specifically, A-sired females had lower methylation levels at CpG site −24 (p<0.05) and
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higher methylation levels at site 141 (p<0.05; Fig. 5C). In the mPFC (Figs. 5B & 5D), there
was a significant main effect of CpG site, F(11, 286)= 14.860, p<0.001, but not Sire or Sex
or their interactions (p’s >0.05). In male offspring sperm, there was a significant main effect
of CpG site, F(11, 198)= 15.261, p<0.001, but not Sire or their interactions (p’s>0.05; see
Supplemental Figure 3).

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4. Discussion
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The results of the present study demonstrate that paternal alcohol exposure confers an
alcohol-resistant phenotype during acquisition for operant alcohol self-administration. A-
sired offspring emit fewer active lever presses and receive fewer alcohol deliveries than
C-sired male and female offspring. Relative to females, differences between male groups
appear earlier, but last for a shorter duration. No sire group differences are seen in
responding for food or water. Additionally, while Bdnf DNA methylation levels are lower
in NAc but greater in mPFC of alcohol-exposed sires, this pattern did not recapitulate in
offspring. Yet, A-sired offspring of both sexes show differential methylation at specific CpG
sites within the Bdnf promoter region. To our knowledge, this is the first study to show
that paternal alcohol exposure decreases operant self-administration during acquisition in
outbred rat offspring of both sexes. Importantly, by employing an operant procedure, results
support that paternal alcohol exposure diminishes alcohol’s reinforcing effects that do not
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reflect impaired learning or performance.

A-sired offspring weighed less in adolescence (PD35) and adulthood (PD75). Consistent
with our results, other rat studies demonstrate intergenerational consequences of paternal
alcohol exposure including lowered body and organ weights 42,43. Yet, alcohol exposure
in mice increases body weights in male offspring 10 perhaps due to species differences.
The lower body weights of A-sired rats in adolescence and adulthood do not appear to
reflect birth factors as there were no sire group differences in body weights at birth nor
were there differences in numbers of litters born or litter size or sex ratio. It is important to
note that the weight difference between offspring at PD35 and PD75 fell into the “small”
effect size category for both sexes, indicating that weight differences between sire groups are
negligible. Further, the lack of group differences in acquisition of food responding suggests
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that body weight likely did not affect operant performance.

Alcohol responding decreased sharply during early training sessions. Specifically, active
lever presses decreased ~60% for males and ~40% for females during the first week of
training. As expected, parallel reductions were also evident for alcohol deliveries. The
higher response levels during the initial training sessions likely reflected the switch from
food reinforcement to alcohol. These patterns of responding were not surprising given that
alcohol is a weaker reinforcer relative to natural rewards, especially in alcohol naïve animals
44. Across sessions, active lever presses were greater than inactive lever presses which were

minimal particularly after the initial alcohol training sessions. This supports that alcohol
was functioning as reinforcer in A-sired and C-sired rats of both sexes. Yet, A-sired rats
pressed less for alcohol and earned fewer deliveries than C-sired rats at the higher alcohol
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concentrations. We saw little effect of sire condition at the lowest concentration (2.5%)
but that may be because it was the initial concentration available. Availability of alcohol
concentrations of 5% and 10% did not alter rate of responding or numbers of reinforcers
earned in male rats. These levels were similar across sessions unlike female rats that pressed
more and earned more reinforcers during the later presentations of the 5% versus the 10%
concentration of alcohol. This sex difference may reflect that the higher level of responding
in female vs. male rats.

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A-sired male mouse offspring display lower preference for low alcohol concentrations
and consume less of moderate concentrations in two-bottle procedures 10,45. A-sired male
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mouse offspring exhibit greater place preference to a low dose of alcohol, while place
aversion occurs at a higher dose that induced a preference in C-sired offspring 11. Our
results corroborate findings of altered sensitivity to rewarding effects of alcohol in male rat
offspring and extend them to females. Offspring of both sexes self-administered less alcohol.
Importantly, results likely do not reflect paternal alcohol-induced learning and memory
impairments as the number of sessions to acquire dipper training, food training, and alcohol
training did not differ. These data support the hypothesis that chronic paternal alcohol
exposure confers a protective effect against the development of addiction-like behaviors for
alcohol in offspring.

Results are inconsistent between preclinical and human studies that show a family history
of alcohol misuse enhances propensity to drink alcohol in children. Children of individuals
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with AUD show lower subjective and behavioral responses to alcohol 2,46 that negatively
correlate with AUD risk 1. This contrasts with the results of the current and prior
rodent studies that report blunted reinforcing effects of alcohol in offspring. Several
factors may be responsible for the lack of agreement. Human studies have not examined
the unique contribution of a father’s alcohol consumption but focused on maternal or
parental (mother + father) alcohol consumption. In rodent studies, alcohol dependence
was induced by maintaining pharmacologically relevant BALs, whereas clinical criteria for
AUD encompasses many other factors. Future preclinical studies could screen sires after
chronic alcohol exposure for behaviors reflective of AUD symptomology in humans perhaps
reconciling these inconsistencies.

Alcohol exposure results in long-lasting changes to Bdnf DNA methylation levels in brain
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reward circuitry of sires, while differential methylation patterns are transmitted to offspring
of both sexes. Specifically, alcohol-exposed sires display lower DNA Bdnf methylation
levels in the NAc and higher levels in the mPFC. We do not find altered Bdnf methylation
patterns in sperm of alcohol-exposed sires or their offspring. BDNF plays a role in
synaptic plasticity and there is a well-established line of research implicating BDNF in
addiction. Related to alcohol, BDNF expression mediates alcohol drinking behaviors in
rodents such that increased expression in reward-circuitry (i.e., ventral tegmental area)
decreases alcohol consumption 48. Thus, epigenetic marks that regulate alcohol-induced
changes in Bdnf expression in sires serve as a promising mechanism by which resilience/
vulnerability to alcohol is passed down to subsequent generations. Indeed, DNA methylation
may recapitulate indirectly via germ line transmission of other epigenetic processes such
as non-coding RNA’s 9. Several CpG sites with differential Bdnf DNA methylation
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levels in offspring encompass transcription factor binding regions that may influence
Bdnf gene expression. A-sired males have altered methylation at CpG sites −11, 43, and
62; the latter two CpG sites are in the Specificity protein 1 (Sp1) and Early growth
response protein 1 (Egr1) transcription factor binding sites (AliBaba2.1), respectfully.
Sp1 transcription factor is involved in cellular processes (e.g., cellular differentiation, cell
growth, apoptosis, immune responses) and chromatin remodeling, specifically in recruiting
histone acetyltransferases 49. The Egr1 transcription factor is involved in brain development,
neural plasticity, and DNA methylation, specifically in DNA demethylation by recruiting the

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Ten-Eleven Translocation 1 protein 50. Altered Bdnf methylation patterns occur in A-sired
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females at CpG sites −24 and 141 (AliBaba2.1), both of which are Sp1 transcription factor
binding sites. Thus, both male and female offspring show differential Bdnf methylation
patterns that, in combination with inherited histone modifications, may contribute to altered
Bdnf signaling.

Previous work in mice shows that alcohol exposure decreases Bdnf methylation in sperm
and in VTA 10, a reward region that contains dopaminergic cell bodies that project to the
NAc and mPFC, in sires. Lower Bdnf methylation levels in VTA are recapitulated in A-sired
male and female offspring 10. Interestingly, this epigenetic profile associates with decreased
alcohol consumption in A-sired male but not female offspring. Similarly, epigenetic-driven
intergenerational changes in BDNF signaling within reward circuitry are reported in other
studies 12,45. The current work differs in several ways, including the use of an outbred rat
strain, duration of paternal alcohol exposure, and timing of mating post-alcohol exposure
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in sires. In the Finegersh and Homanics study 10, mice were mated immediately following
the last alcohol vapor session, whereas in our study, males were mated 8 weeks after
alcohol exposure. Thus, our results extend previous work by demonstrating that paternal
alcohol exposure several weeks prior to conception has epigenetic consequences leading
to decreased reinforcing effects of alcohol in both male and female offspring. It would be
advantageous in the future to examine sensitivity to alcohol in offspring of sires that actively
self-administered alcohol.

While the current study has several strengths, including the use of both sexes and
employment of operant procedures, there are some limitations. Food restriction may
cause caloric compensation wherein alcohol’s caloric value maintained operant responding.
Although both C-sired and A-sired offspring were food restricted to the same extent, A-sired
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offspring, which weighed less than C-sired offspring, may have been more impacted by
alcohol during earlier training sessions. However, this effect may have subsided as both
groups were responding at equivalent levels during the final week of self-administration.
Nonetheless, progressive ratio schedules, which provide an index of drug and alcohol
motivation, would be useful in determining the reinforcing effects of alcohol in the different
groups. In addition, while paternal alcohol exposure results in several changes in Bdnf DNA
methylation levels, transcriptional and translational activity of the gene were not measured.
Lastly, we did not directly examine potential changes in maternal behavior in dams that were
mated to alcohol-exposed sires. This may be an important confound considering that dams
can sense the fitness of their mates and alter their care of pups accordingly 51.

In summary, paternal alcohol exposure decreases alcohol self-administration during

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acquisition particularly at higher concentrations (5%−10%). Sex differences occur in the

timing of these effects. Yet, by the end of the acquisition period, sire groups of both sexes
respond similarly for 5% alcohol suggesting that long-term drinking history may offset
the protective effect of paternal alcohol exposure. Similar to previous rat studies, A-sired
offspring had lower body weights during adolescence and adulthood and yet, no group
differences are seen in responding for food or water. Additionally, alcohol differentially
altered Bdnf DNA methylation levels in both the NAc and mPFC of sires several weeks
after their last exposure. Aberrant DNA methylation patterns are also seen in the NAc, but

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not the mPFC, of male and female A-sired offspring. Taken together, our findings support
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the hypothesis that paternal alcohol exposure has intergenerational consequences, including
decreasing the initial reinforcing effects of alcohol in offspring that may be a result of
transmitted epigenetic marks. Future research should investigate whether paternal alcohol
exposure alters operant responding for alcohol under maintenance conditions or if it affects
the ability of alcohol or other cues to reinstate extinguished responding for alcohol. Such
research would address issues related to chronic alcohol use and the ability to abstain from
use after cessation.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
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Funding and Disclosure

This work was supported by the National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism
(AA013476, TAK; AA026495, SJN) and the Toomim Family Fund (DAN). The authors declare no competing
financial interests in relation to the work described in this study.

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Fig 1.
Experimental Timeline. Sires were exposed to chronic intermittent ethanol in vapor
chambers (16 h/day; 5 days/week) or to air for 6 weeks. Eight weeks later, rats were
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mated with alcohol-naive females. At postnatal day 75, alcohol- and control-sired F1
offspring began training to drink water from operant dipper (weeks 1–2) followed by
food training (weeks 3–6). After food training, alcohol- and control-sired offspring were
assessed in acquisition of alcohol self-administration (weeks 7–14) in which increasing
alcohol concentrations (2.5%, 5%, & 10%, v/v). were delivered after one lever press (fixed
ratio 1). DNA methylation levels of the brain derived neurotrophic factor (Bdnf) gene were
measured in sperm, nucleus accumbens (NAc), and medial prefrontal cortex (mPFC) in sires
and in offspring.
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Fig 2.
Active lever presses for control- (C-sired; open circles) and alcohol-sired (A-sired; filled
circles) offspring during acquisition of alcohol self-administration under a fixed ratio 1
(FR1) schedule of reinforcement. Active lever presses are presented as mean (± SEM) for
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males (A) and females (C) for each day. Daily active lever presses by week are presented
as mean (± SEM) for males (B) and females (D). An asterisk (*) represents a significant
difference between A and C-sired offspring (p < 0.05).
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Fig 3.
Alcohol deliveries for control- (C-sired; open circles) and alcohol-sired (A-sired; filled
circles) offspring during acquisition of alcohol self-administration under a fixed ratio 1
(FR1) schedule of reinforcement. Alcohol deliveries are presented as mean (± SEM) for
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males (A) and females (C) for each day. Daily alcohol deliveries by week are presented
as mean (± SEM) for males (B) and females (D). An asterisk (*) represents a significant
difference between A- and C-sired offspring (p < 0.05).
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Fig 4.
Bdnf DNA methylation levels for control- (open bars) and alcohol-treated (filled bars) sires.
Percentage of DNA methylation at CpG sites are presented as mean (± SEM) within the
nucleus accumbens (A) and medial prefrontal cortex (mPFC) (C) of sires. Percentage of
methylation levels across CpG sites are presented as mean (± SEM) within the nucleus
accumbens (B) and mPFC (D). An asterisk (*) represents a significant difference between
control and alcohol-treated sires (p< 0.05). ** indicates sire differences at p<0.01.
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Fig 5.
Bdnf DNA methylation levels for control- (C-sired; open bars) and alcohol-sired (A-sired;
filled bars) offspring. Percentage of DNA methylation at CpG sites are presented as mean
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(± SEM) within the nucleus accumbens of male (A) and female offspring (C). Percentage of
DNA methylation at CpG sites are presented as mean (± SEM) within the medial prefrontal
cortex (mPFC) of male (B) and female (D) offspring. An asterisk (*) represents a significant
difference between C- and A-sired offspring (p < 0.05). ** indicates a significant difference
between C- and A-sired offspring at p<0.01.
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Table 1.

Litter characteristics for alcohol and sires

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Control sires Alcohol sires

Number of litters 10 10

Mean pups/litter (standard error) 12.4 (1.3) 10.5 (1.8)

Number of male offspring 52 46

Number of female offspring 43 49

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Table 2.

Body weights of offspring

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Sex Group Body Weights (Mean ± SEM g)

PD 1 PD 4 PD 7 PD 10 a a
PD 35 PD 75
Male C-sired 8.0 ± .22 12.7 ± .08 16.9 ± .46 26.2 ± .90 147.5 ± 3.3 425.4 ± 5.1
A-sired 6.7 ± .11 10.5 ± .16 17.3 ± .39 23.9 ± .39 b b
126.9 ± 7.1 387.3 ± 8.5
Female C-sired 8.2 ± .31 12.1 ± .17 16.5 ± .31 23.8 ± .55 125.0 ± 2.1 241.5 ± 2.7
A-sired 6.8 ± .13 10.2 ± .15 16.7 ± .30 23.2 ± .37 b b
108.4 ± 5.0 224.6 ± 4.1

Note. C-sired, control-sired: A-sired, alcohol-sired; PD, postnatal day;

a
indicates sex differences at p<0.05;
b
indicates sire differences at p<0.001
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Table 3.

Sessions to reach acquisition criteria for operant alcohol self-administration

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Sessions to acquisition (Mean ± SEM)

Sex Group Water training Food training Alcohol training

Male C-sired 7.3 ± 4.4 13.4 ± 4.3 9.9 ± 2.8
A-sired 6.9 ± 2.8 16.8 ± 2.9 7.7 ± 2.5
Female C-sired 6.2 ± 3.7 17.2 ± 3.8 6.4 ± 2.5
A-sired 5.3 ± 3.1 14.5 ± 2.1 6.9 ± 2.4
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