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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Alcohol. 2018 Dec 29;79:59–69. doi: 10.1016/j.alcohol.2018.12.004

PRENATAL ETHANOL EXPOSURE ATTENUATES SENSITIVITY TO THE AVERSIVE EFFECTS OF ETHANOL IN ADOLESCENCE AND INCREASES ADULT PREFERENCE FOR A 5% ETHANOL SOLUTION IN MALES, BUT NOT FEMALES

Jonathan Kent Gore-Langton 1, Linda Patia Spear 1
PMCID: PMC6692249  NIHMSID: NIHMS1517615  PMID: 30597200

Introduction

Prenatal alcohol exposure has been shown to be an important predictor of later alcohol use in human adolescents (Baer et al., 1998 & 2003). Likewise, prenatal or neonatal ethanol exposure has been reported to elevate ethanol intake in adolescent rats, even following exposure to doses well below those necessary to induce neuroteratogenic or morphological deficits (Abate et al., 2008; Fabio et al., 2013 & 2015). Even brief exposure to a moderate dose of ethanol from gestational day (G) 17–20 has been shown to enhance ethanol intake (Chotro & Arias, 2003) and prevent expression of conditioned place aversions (Pautassi et al. 2012) in infant rats. One mechanism through which prenatal ethanol exposure may increase ethanol intake during adolescence is an exaggeration of the normal adolescent insensitivity to the aversive effects of ethanol (Anderson et al., 2008; Fabio et al., 2015; Saalfield & Spear, 2016). Indeed, a meta-analysis of rodent genetic studies found that sensitivity to the aversive effects of ethanol (assessed via conditioned taste aversion [CTA]) plays an important role in moderating ethanol intake and is more strongly correlated with ethanol intake than sensitivity to the rewarding properties of ethanol (Green & Grahame, 2008).

CTA is a classical conditioning paradigm commonly used to assess the aversive effects of alcohol and other drugs. During conditioning, a novel tastant (conditioned stimulus - CS) is paired on one or more occasions with injection of a malaise-inducing drug (unconditioned stimulus – US). To the extent that the animal associates the aversive effects of the US with the CS, consumption of the CS should be attenuated on subsequent exposures. Relative to adults, adolescents are less sensitive to this effect of ethanol, requiring a higher dose or more pairings of the drug with a novel tastant to develop a CTA (Anderson et al., 2010). This age-related insensitivity to ethanol-induced CTA may permit greater ethanol intake in adolescents relative to adults, with adolescent rodents often voluntarily consuming 2 to 3-fold greater amounts of ethanol per occasion relative to adults (Brunell & Spear, 2005; Doremus et al., 2005; Vetter et al., 2007). Indeed, a study by Schramm-Sapyta et al. (2010) using male rats from an outbred Sprague-Dawley derived strain found that CTA was inversely correlated with post-deprivation consumption of an 8% ethanol solution in adolescents, but not adults.

While there is mounting evidence from rodent models that prenatal ethanol exposure can increase ethanol intake in adolescence, a time when alcohol use is typically initiated in humans (Miller & Spear, 2006), contributors to this later elevation in intake are still being explored. Factors suggested to contribute to this elevation include altered orosensory properties that enhance the palatability of ethanol (Chotro & Arias, 2003; Arias & Chotro, 2005a, b; Youngentob & Glendinning, 2009), induction of social anxiety that can be alleviated by ethanol exposure (Mooney & Varlinskaya, 2018), or exaggeration of normal adolescent insensitivities to the aversive effects of ethanol. The effect of prenatal ethanol exposure combined with the adolescent-typical insensitivity to the aversive effects of ethanol could create a “double hit” (Eade et al., 2009), elevating adolescent consumption and increasing the probability for the development of alcohol use disorders (AUDs) which persist into adulthood. In humans, prenatal alcohol exposure has shown to be more predictive of adolescent and adult alcohol use than a family history of alcohol problems (Baer et al., 1998 & 2003). Given that an important risk factor for AUD among adults is their pattern of consumption during adolescence (Grizenko & Fisher, 1992), it is important to understand the mechanisms through which prenatal ethanol exposure affects this pattern of drinking.

The aims of this study were two-fold. First, to determine if exposure to a moderate dose (2g/kg) of ethanol during late gestation (G17–20) would attenuate sensitivity to the aversive effects of ethanol in adolescent rats assessed via a conditioned taste aversion (CTA) paradigm. The second aim of the study was to determine whether prenatal ethanol exposure would increase consumption of and preference for ethanol intake in late adolescence and young adult rats using a 3-bottle choice paradigm. The drinking paradigm of 2 g/kg from G17–20 was chosen based on previous studies that have found enhanced postnatal ethanol intake using a low or moderate dose during this period, such as Arias & Chotro (2005a) and Fabio et al. (2013). Moreover, evidence suggests that during late gestation the fetus can sense the chemosensory and toxic characteristics of ethanol (Abate et al., 2001; Chotro & Arias, 2003), which may modify CTA sensitivity to ethanol in adolescence.

General Methods

Subjects

Sprague-Dawley male and female rats bred and reared in our colony at Binghamton University were used in this study. The day after birth, all litters were culled to 8–10 pups and housed with their dams until weaning on postnatal day (P) 21. At weaning, each animal was pair-housed with a same sex non-littermate from the same prenatal exposure (ethanol, water, non-manipulated) group, with housing pairs not differing in age by more than 1 postnatal day. Animals were maintained in a temperature controlled (20–22°C) vivarium on a 12-/12-h light/dark cycle (lights on at 7:00) with ad libitum access to food (Purina Rat Chow, Lowell, MA) and tap water. All procedures were conducted in accord with guidelines established by the National Institutes of Health using protocols approved by the Binghamton University Institutional Animal Care and Use Committee.

Breeding and Prenatal Exposure

Mating occurred over 4–5-day periods, during which time 2 females were placed in a single cage with one male. During this period females were vaginally lavaged daily between 9:00–11:00 until sperm were detected, with this day defined as gestational day 0. Following mating, each pair of females remained housed together for 2 weeks prior to being isolate housed. From G17–20, dams were given once daily intragastric intubations (i.g.) of 2.0 g/kg ethanol (20% v/v) or water vehicle (isovolumetric to the ethanol solution) between 9:00–11:00 hours. Non-manipulated dams were not intragastrically intubated and were only exposed to standard animal facility housing and rearing conditions.

Experiment 1: Effect of Prenatal Ethanol Exposure on Sensitivity to the Aversive Effects of Ethanol in Adolescence

The design of this experiment was a 3-prenatal Exposure condition (ethanol, water, or non-manipulated) X 2 Sex (male, female) X 4 Dose (0, 1, 1.25 & 1.5 g/kg ethanol) factorial. Given that the nature of the conditioned stimulus (CS) can affect conditioning (Risinger & Cunningham, 1995, 2000), two different tastants were used as the CS in separate groups of animals: Experiment 1.1: supersaccharin [(3% sucrose, 0.125% saccharin in water; modified from Ji et al., 2008; see Morales et al., 2014); Experiment 1.2: sodium chloride (0.9% NaCl)]. Beginning on P27 (± 1 day) each pair of animals was 50% water restricted as described below. Twenty-four hours after the onset of water restriction, the first CTA session occurred. At the onset of each session, animals were weighed; each housing pair was then separated in a novel test cage with a wire-mesh divider and placed in a room containing a white noise generator and dim light (15–20 lux). Fifteen mins later, the conditioning session began. At the onset of conditioning, each animal was provided with one bottle containing the CS for a 30 min access period. Immediately thereafter, the bottle was removed, and the animal was injected with the designated ethanol dose via intraperitoneal (i.p.) administration of 0 (0.9% saline solution), 1, 1.25, or 1.5 g/kg of 20% ethanol in physiological saline. Control animals in this experiment were injected with 0.9% saline isovolumetric to 2.25 g/kg. This control volume was utilized because the initial squads of animals examined doses of 1.5, 1.75, 2 and 2.25 g/kg and observed CTA in all exposure groups even at 1.5 g/kg, leading to downward adjustment of ethanol doses for this study. Since the saline animals used with the initial dose range received volumes isovolumetric to the 2.25 g/kg dose, this volume was maintained throughout the experiments.

Intake in the CTA experiment was assessed by weighing bottles before and after each intake session. Each housing pair received the same drug challenge and remained separated for an additional 15 mins following the 30 min access period. Animals were then returned to their home cages and provided ad libitum food and water for the next 24 hours. One day prior to each CTA conditioning and test session animals were given 50% of the water they consumed in their home cage within the past 24 h. This cycle of 50% water deprivation followed by 30 min of solution access, injection of the same drug solution as on day 1, and 24 hrs. of ad libitum food and water access continued for 3 more access sessions, with no injection administered after the final session. This procedure allowed tracking of the emergence of CTA over days.

Experiment 2: Effect of Prenatal Ethanol Exposure on Voluntary Ethanol Intake

2.1. Late Adolescence

The design of this experiment was a 2 prenatal Exposure (ethanol or water) X 2 Sex (male or female) X 3 Solution (10% ethanol, 5% ethanol & Vehicle) factorial. As the most pronounced differences in CTA sensitivity were found among ethanol and water exposed animals in Experiment 1, only these two exposure groups were examined in Experiments 2.1 and 2.2. Beginning on P35 (± 1 day), adolescent animals that had not undergone CTA were given 18 hr. access to 3 bottles containing 10% ethanol, 5% ethanol and water beginning 15 min following the start of the dark cycle (i.e., from 19:15 hrs. - 13:15 hrs.). These bottles were presented every other day in the animals’ home cages for 3 days per week (with 1 day off between days and 2 days off before the start of a new cycle (e.g. M, W, F) for 3 weeks. A sucrose fading procedure adapted from Fabio et al. (2015) was used in which 1% sucrose was mixed in all solutions during the first week (sessions 1–3), 0.5% sucrose was used during the second week (sessions 4–6) and the solutions were prepared in water (i.e., 0% sucrose) during the final week (session 7–10). The position of the three bottles was counter-balanced across conditioning sessions to avoid place biases. On the 10th and final session (that occurred 2 days after the 9th session), bottles were placed on cages for 30 min and animals rapidly decapitated thereafter for collection of trunk bloods for analysis of blood ethanol concentrations (BECs).

2.2. Emerging to Early Adulthood

Animals in experiment 2.2 were exposed to the same procedures and used the same experimental design as experiment 2.1, except that the start of intake testing did not begin until P56–60.

Data Analyses:

To determine whether prenatal exposure condition significantly affected adolescent growth, body weights from Experiment 1 were analyzed over Days using separate 3 X 4 mixed model Analyses of Variance (ANOVAs) for males and females, each with a between-subjects factor of Exposure (ethanol, water and non-manipulated) and a within-subjects factor of Day (1–4), with a similar 2 Exposure X 4 Day ANOVA used for the body weight data of Experiment 2. In the case of an interaction between Exposure and Day, differences between groups were tested using one-way ANOVAs on each Day, with Fisher’s LSD tests used to determine the locus of significant differences in this and all subsequent ANOVAs. For the CTA data in experiments 1.1–1.2 we had considered using Dunnett’s post-hoc tests, however this was not a possibility given that there was not a single control group, but rather a separate control group for each exposure (ethanol, water, non-manipulated) to which all dose comparisons were made. For all analyses, an ANOVA F-test was first performed and rejected before any pairs of means were compared. By adding this requirement, the overall error rate of Fisher’s LSD was maintained close to α. All findings were considered significant at p< 0.05.

In Experiment 1, animals that consumed less than 0.5 grams of the CS on the first CTA conditioning day were excluded from analysis. Among males, 3% of the ethanol, 2% of the water and 2% of the non-manipulated animals were dropped due to low consumption; corresponding percentages for females were 7%, 0% and 0%, respectively. Pearson chi-square tests conducted using a 2-intake level (low vs. normal) X 3 Exposure (ethanol, water, non-manipulated) design revealed no significant relationship between the number of low drinking animals and exposure groups in males, whereas there was a significant relationship in females χ2= (1, N=237) = 10.97, p<.01, with a larger proportion of low drinkers in the ethanol group. To determine if there were differences in pre-conditioning consumption of the tastant across groups after the low consuming (≤0.5 g) animals were excluded, baseline intake of NaCl and supersaccharin were analyzed separately for each Sex using a one-way ANOVA for Exposure (ethanol, water, non-manipulated).

Due to significant differences in baseline CS intake across groups (i.e., see S1), CTA test data for each Exposure X Dose group were converted into a percentage of the corresponding saline group’s (e.g., ethanol-saline, water-saline, or non-manipulated- saline) mean consumption for that day. These data were then averaged and a significant decrease relative to saline controls was considered development of CTA. These CTA data were then analyzed across test days (Days 2–4), using separate ANOVAs for males and females, each with between subjects factors of Exposure (ethanol, water and non-manipulated) and Dose (0, 1, 1.25 and 1.5 g/kg) and a within subjects factor of Day (2–4).

For analysis of the voluntary intake data in adolescent and adults in Experiments 2.1 and 2.2, 3-Way ANOVAs using a between subjects factor of Exposure (ethanol, water) and within subjects factors of Solution (10% ethanol, 5% ethanol) and Week (1–3) were used to examine both intake of ethanol in g/kg and % preference for each ethanol solution in each experiment. For the g/kg intake analyses, average ethanol consumed weekly of each solution by each animal was converted into grams ethanol/kilograms of body weight. Likewise, weekly % preference for each of the ethanol solutions was calculated as the total amount of the solution consumed divided by the total amount of fluid consumed in grams over the 3 sessions multiplied by 100. In cases where consumption on a session could not be accurately measured due to bottle leakage or blockade, g/kg intake and percent preference for that week were estimated using the average of the remaining sessions for that week for each animal

On the 10th and final access session, the total amount of ethanol (g/kg) that each exposure group drank was calculated as the sum of g/kg intake of the 5% and 10% ethanol solutions divided by the animal’s body weight in kg. Pearson product-moment correlations were conducted separately for each Sex and Exposure group (ethanol and water) to investigate the relationship between total amount of ethanol consumed and BECs on the 10th session. One-way ANOVAs conducted within each Sex were used to compare the prenatal groups in terms of BECs as well as the total amount of ethanol consumed on the 10th session. BECs were determined by the collection of trunk bloods immediately following the 30 min drinking session. Samples were then frozen and stored at −80°C until assessed using a Hewlett Pack ard 5890 series II Gas Chromatograph (Wilmington, DE) (see Ramirez & Spear, 2010, for further details).

Results

Experiment 1.1 Supersaccharin CTA

Body weight.

Baseline body weight data are summarized in Table 1 for all experiments. Among males in Experiment 1.1, there was a main effect of Exposure [F(2,137)=3.28, p<.05], with post-hoc tests revealing that the ethanol group weighed significantly less than water animals. There was also a main effect of Exposure in females [F(2,128)=3.03, p=.05], with Fisher’s post-hocs revealing that non-manipulated animals weighed significantly less than those in the water group.

Table 1.

Baseline Body weights at the start of each experiment. Means presented as mean (± SEM).

Experiment Procedure Baseline Day Sex Exposure (n) Body Weight (g)
1.1 CTA (Tastant: SS) P28 (± 1) Males Ethanol (52) 100.3 (± 1.7) *
Water (49) 105.5 (± 1.4)
Non-manipulated (39) 104.4 (± 1.6)
Females Ethanol (50) 94.3 (± 1.4)
Water (45) 97.6 (± 1.3)
Non-manipulated (35) 92.7 (± 1.5) @
1.2 CTA (Tastant: NaCl) Males Ethanol (45) 101.0 (± 1.5) *
Water (35) 110.3 (± 1.7)
Non-manipulated (31) 101.7 (± 1.3) @
Females Ethanol (37) 91.3 (± 1.7) *
Water (27) 99.0 (± 1.4)
Non-manipulated (34) 91.8 (± 1.1) @
2.1 Voluntary Drinking (Adolescent) P35 (± 1) Males Ethanol (8) 184.0 (± 4.4)
Water (6) 192.4 (± 5.7)
Females Ethanol (8) 157.4 (± 4.2)
Water (6) 158.3 (± 2)
2.2 Voluntary Drinking (Adults) P56–60 Males Ethanol (8) 345.9 (± 11.9)
Water (10) 324.8 (± 6.6)
Females Ethanol (8) 227.6 (± 6.9)
Water (12) 212.5 (± 4.3)
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*

ethanol < water

@

non-manipulated < water

Among animals in experiment 1.1 and 1.2, ethanol and non-manipulated animals tended to have lower baseline bodyweights than water exposed animals. There were no differences in baseline bodyweights among ethanol and water exposed groups in experiment 2.

Baseline intake.

The one-way ANOVA revealed a main effect of Exposure [F(2,128) =4.46, p< .05] in females, but not males. Fisher’s post-hocs showed that females prenatally exposed to water consumed significantly less supersaccharin in grams/kilogram (g/kg) at baseline (41.98 ± 2.96) compared to both ethanol (54.25 ± 2.45) and non-manipulated (49.95 ± 3.98) females. In males, the baseline SS intake in g/kg for ethanol exposed animals was (55.59 ± 2.17), for water exposed (52.21 ± 2.61), and for non-manipulated (49.59 ± 3.8). Due to these sex-related baseline differences, all subsequent analyses of CTA data focused on analyses based on % saline within each sex.

Conditioned Taste Aversion.

Analysis of the % saline data in adolescent males revealed that those prenatally exposed to ethanol showed attenuated sensitivity to ethanol induced CTA relative to their control counterparts. The ANOVA of these data revealed a main effect of Dose [F(3,128)=37.72, p<.01], Day [F(2,256)=15.82, p<.01] as well as interactions of Exposure X Dose [F(6,128)=2.68, p<.05] and Day X Dose [F(6,256)=3.04, p<.01]. Fisher’s LSD tests on data collapsed across Day to explore the Exposure X Dose interaction revealed significant CTA at doses of 1 g/kg and higher in the water group, at doses of 1.25 g/kg and 1.5 g/kg in the non-manipulated group, but only at the 1.5 g/kg dose in the ethanol group (see Fig. 1). The Day X Dose interaction revealed that CTA emerged by the third conditioning day at the 1 g/kg dose and by the second conditioning day at the 1.25 and 1.5 g/kg doses. Among females, there was a main effect of Dose [F(3,114)=32.18, p<.01], Day [F(2,228)=17.71, p<.01], and interactions of Day X Dose [F(6,228)=2.46, p<.01] and Day X Exposure [F(4,228)=2.46, p<.05]. Fisher’s post-hocs on the Day X Exposure interaction, however, did not reveal significant differences in consumption between exposure groups on any day. Post-hocs on the Day X Dose interaction revealed that there were significant decreases in consumption relative to saline at the 1 g/kg dose by day four and at the 1.25 and 1.5 g/kg doses by day two.

Fig. 1. Experiment 1.1 Supersacharin CTA:

Fig. 1.

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(a) Adolescent males in the ethanol group had an attenuated sensitivity to ethanol CTA compared to both the water and non-manipulated groups. CTA was determined as a significant reduction in consumption for each Exposure X Dose group relative to their own saline controls. (b) In adolescent females there were no differences in effective CTA inducing dose among ethanol, water and non-manipulated groups. Total n for experiment 1.1 = 270 derived from 46 litters.

& Denotes significant CTA for ethanol group

# Denotes significant CTA for water group

* Denotes significant CTA for non-manipulated group

Experiment 1.2 Sodium chloride CTA

Body weight.

In the ANOVAs of body weights in both males and females, there was a main effect of Exposure [F(2,96)=8.18, p<.01, F(2,119)=11.22, p<.01, respectively], with Fisher’s post-hoc tests showing that in animals of both sexes, ethanol and non-manipulated animals weighed significantly less than the water group.

Baseline intake.

Baseline NaCl intake measured in g/kg of body weight was not affected by prenatal exposure in either sex (see S1). In females, the baseline NaCl intake in g/kg was (89.82 ± 5.45) for ethanol exposed, (80.91 ± 12.88) for water exposed, and (81.69 ± 5.89) for non-manipulated animals. In males, the baseline NaCl intake in g/kg was (77.36 ± 5.57) for ethanol exposed, (78.09 ± 4.93) for water exposed and (85.18 ± 3.8) for non-manipulated animals.

Conditioned taste aversion.

The ANOVA of % saline consumption on test days (Days 2–4) in males revealed no effects of Exposure, although there was a main effect of Dose [F(3,108)=11.25, p<.01], Day [F(2,216)=23.18, p<.01] and a Day X Dose interaction [F(6,216)= 3.25, p<.01]. Fisher’s post-hocs on data collapsed across exposure condition to examine this interaction revealed that the 1.25 g/kg dose induced CTA by the third conditioning session and the 1.5 g/kg dose by the second conditioning session (Fig. 2a). In females, there was a main effect of Exposure [F(2,83)= 5.26, p<.01], Dose [F(3,83)=7.03, p<.001] and an interaction of Day X Exposure [F(4,166)=3.19, p<.05). Fisher’s post-hocs on data collapsed across CTA Dose to examine this interaction revealed that compared to the ethanol females, the water and non-manipulated females consumed significantly less of the CS on Days 2–3 and 3–4, respectively (Fig.2b). While these data provide some evidence for attenuated CTA after prenatal ethanol exposure in females, the absence of an interaction involving Dose requires that this conclusion be tempered accordingly.

Fig. 2. Experiment 1.2 Sodium Chloride CTA:

Fig. 2.

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(a) Among males, there were no differences in consumption of NaCl between Exposure groups on the same Day. (b) Among females, the water and non-manipulated groups consumed significantly less NaCl relative to their own control groups on Days 2–3 and 4, respectively compared to the ethanol group. Data are presented collapsed over Dose. Total n for experiment 1.2 = 209 derived from 41 litters.

* Denotes significant difference between ethanol and water.

# Denotes significant difference between ethanol and non-manipulated.

Experiment 2 Voluntary Ethanol Intake-3 bottle test

2.1. Adolescents

Body weight.

In the body weight analyses prior to the first drinking day during adolescence, there was no effect of prenatal Exposure (ethanol or water) on baseline body weight in grams (see Table 1).

Percent preference data and grams/kilogram intake data.

Among males, no effects of prenatal ethanol exposure on adolescent ethanol intake were observed (see Fig. 3a). A 3-Way ANOVA 2 (Exposure) X 2 (Solution) X 3 (Week) examining the % preference data revealed only a main effect of Solution [F(1,12)= 10.31, p<.01] and an interaction of Solution X Week [F(2,24)=3.63, p<.05], with a greater preference for the 5% than 10% ethanol solution on Weeks 2 and 3 (see Table 3). A similar ANOVA on g/kg intake revealed interactions of Exposure X Week [F(2,24)=3.35, p=.05] and Solution X Week [F(2,24)=3.77, p<.05], although Fisher’s post-hocs examining each of these interactions did not reveal significant differences in consumption on any of the weeks between exposure groups or between the two ethanol solutions.

Fig.3. Experiment 2.1 Voluntary Drinking Adolescents:

Fig.3.

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(a) Among males, there were no differences in % preference or g/kg intake for either the 5% or 10% ethanol solutions between exposure groups. (b) In females, ethanol-exposed animals showed significantly lower preference and g/kg intake for the 5% ethanol solution compared to water exposed animals. There was no difference in preference or intake for the 10% ethanol solution between exposure groups. Total n for experiment 2.1= 28 animals derived from 20 litters (same litters as used in experiment 2.1)

*= Significant difference between ethanol and water pre-exposure groups

Table 3:

Weekly percent preferences for 5% and 10% ethanol

Age Sex Exposure Week 1 Week 2 Week 3
5% 10% 5% 10% 5% 10%
Adolescents Male Ethanol 24.00 (± 6.52) 19.87 (± 6.54) 32.12 (± 6.30) 13.27 (± 2.44) 34.42 (± 6.03) 12.76 (± 3.96)
Water 24.75 (± 8.38) 13.58 (± 4.78) 41.12 (± 8.32) 15.37 (± 4.54) 45.81 (± 11.12) 21.21 (± 7.93)
Female Ethanol 19.39 (± 5.41) 22.41 (± 4.74) 38.05 (± 7.82) 15.24 (± 2.80) 37.37 (± 7.79) 20.03 (± 4.59)
Water 49.22 (± 7.23) 17.93 (± 3.04) 56.82 (± 10.49) 11.59 (± 1.98) 54.51 (± 9.24) 11.11 (± 0.95)
Adults Male Ethanol 30.97 (± 4.70) 6.17 (± 0.67) 58.19 (± 8.36) 5.82 (± 0.58) 61.59 (± 7.70) 7.78 (± 0.77)
Water 25.54 (± 8.08) 12.37 (± 4.13) 26.48 (± 5.18) 18.74 (± 4.60) 41.58 (± 9.17) 18.15 (± 5.03)
Female Ethanol 26.13 (± 4.28) 17.05 (± 7.20) 35.70 (± 9.46) 13.37 (± 4.02) 43.37 (± 8.98) 15.00 (± 2.38)
Water 33.01 (± 4.97) 10.28 (± 2.81) 48.51 (± 5.88) 8.81 (± 1.17) 56.88 (± 5.75) 10.97 (± 1.45)
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Means and SEMS of percent preference for the 5% and 10% ethanol solutions are shown for Age (adolescent,adult), Sex (male,female) and Exposure group (ethanol,water) across all weeks.

Among females, consumption of the 5% ethanol solution was lower in prenatal ethanol-exposed than water-exposed females, as can be seen in Fig. 3b. A 2 (Exposure) X 2 (Solution) X 3 (Week) ANOVA comparing percent preference found a main effect of Solution [F(1,12)= 40.31, p<.01], and interactions of Exposure X Solution [F(1,12)= 11.2, p<.01] and Solution X Week [F(2,24)=4.23, p<.05]. Fisher’s post-hocs on the Exposure X Solution interaction showed that the water exposed females preferred the 5% ethanol solution significantly more than the ethanol-exposed females. Post-hocs on the Solution X Week interaction showed that the 5% ethanol solution was significantly preferred relative to the 10% ethanol solution on each week (Table 3). A similar ANOVA of ethanol intake in g/kg revealed a main effect of Week [F(2,24)=4.19, p<.05] and interactions of Exposure X Solution [F(1,12)=11.09, p<.01] and Solution X Week [F(2,24)=5.63, p<.01]. Fisher’s post-hoc analysis of the Exposure X Solution showed that water exposed females consumed significantly more of the 5% ethanol solution than ethanol-exposed females, with intake of the 10% ethanol solution not differing between exposure groups (see Fig. 3b). Post-hocs on the Solution X Week interaction revealed that g/kg intake of the 5% ethanol was significantly greater than 10% ethanol on week two [5% ethanol: (3.87± 0.61), 10% ethanol: (2.37 ± 0.33)].

2.2. Adults

Body weight.

In the analyses of body weight prior to the onset of voluntary drinking in adulthood, there was no effect of Exposure (ethanol or water) in either males or females (see Table 1).

Percent preference and grams per kilogram consumption.

In adult males, a 2 (Exposure) X 2 (Solution) X 3 (Week) ANOVA examining percent preference revealed main effects of Solution (F[1,16]= 41.75, p<.01) and Week (F[2,32]=15.83, p<.01), with these main effects tempered by interactions of Solution X Exposure (F[1,16]=15.83, p<.01) and Solution X Week (F[2,32]=3.45, p<.05). Fisher’s post-hocs on the Solution X Exposure interaction showed significantly greater preference for the 5% ethanol solution among the ethanol-exposed males, but no difference in preference for the 10% ethanol solution between Exposure groups (see Fig. 4a). Post-hocs on the Solution X Week interaction revealed that for all weeks there was a significantly greater preference for the 5% compared to the 10% ethanol solution (see Table 3). A similar ANOVA on g/kg intake revealed a main effect of Solution (F[1,16]=9.01, p<.01), Week (F[2,32]=5.04, p<.05) and an interaction of Solution X Exposure (F[1,16]=11.84, p<.01). Fisher’s post-hocs on the interaction of Solution X Exposure revealed that ethanol-exposed males consumed significantly more of the 5% ethanol solution while water exposed animals consumed significantly more of the 10% ethanol solution. The main effect of Week reflected an increase in total ethanol intake from weeks 1 to 2 and 3.

Fig. 4. Experiment 2.2 Voluntary Drinking Adults:

Fig. 4.

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(a) Among males, prenatal ethanol exposed animals showed significantly greater preference and g/kg intake of the 5% ethanol solution relative to water exposed males, while also consuming significantly less in g/kg of 10% ethanol compared to water exposed males. (b) Adult females that were pre-exposed to ethanol showed significantly less preference and intake of the 5% ethanol solution relative to water exposed females. Total n for experiment 2.2 = 34 animals derived from 20 litters (same litters as used in experiment 2.1)

* Denotes significant difference between ethanol and water groups

Adult females exposed prenatally to ethanol, like their adolescent counterparts, exhibited lower intake of the 5% ethanol solution than did water-exposed females (see Fig.4b). A 2 (Exposure) X 2 (Solution) X 3 (Week) ANOVA of the % preference data showed a main effect of Solution (F[1,18]=65.67, p<.01) and Week (F[2,36]=7.53, p<.01) as well as interactions of Exposure X Solution (F[1,18]=5.48), p<.05) and Solution X Week (F[2,36]=4.92, p<.05). Fisher’s post-hocs on the Exposure X Solution interaction revealed that the water exposed females had a significantly greater preference for the 5% ethanol solution than ethanol-exposed females, whereas the exposure groups did not differ in their preference for the 10% ethanol solution. Post-hocs conducted on the Solution X Week interaction revealed that intakes of the 5% ethanol solution increased over weeks, although the 5% ethanol solution was significantly more preferred than the 10% ethanol solution each week (see Table 3). A similar ANOVA examining g/kg intake in the adult females showed a main effect of Solution (F[1,18]=12.20, p<.01) and an interaction of Exposure X Solution (F[1,18]= 5.43, p<.05). Fisher’s post-hocs on the Exposure X Solution interaction revealed that the water exposed females consumed significantly more of the 5% ethanol solution than ethanol-exposed females. However, no differences in consumption of the 10% ethanol solution emerged between exposure groups, or between solutions within the ethanol-exposed females.

Thus, preference and intake of a 5% ethanol solution was increased in adult males exposed gestationally to ethanol, but decreased in their female counterparts.

BECs.

Total ethanol consumption (g/kg) and BEC data from the 10th access session in adults revealed BECs that were generally in the low to moderate range when taken immediately after the 30 min access period on this day (see Fig.5). Ethanol consumption and BECs were significantly correlated in all groups, except for the ethanol pre-exposed females where a trend was observed. One-way ANOVAs conducted separately for each sex comparing BECs between exposure groups (ethanol vs. water) as well as total amount of ethanol consumed in g/kg on this consumption day revealed no significant exposure group differences in BECs or ethanol consumed, despite a trend for water pre-exposed females to drink significantly more total ethanol in g/kg than ethanol pre-exposed females (see Table 2).

Fig. 5.

Fig. 5.

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In adults, that underwent voluntary drinking, a significant correlation was found between ethanol intake and BECs on the final 30 min intake session in all exposure and sex groups, except for the ethanol females where only a trend was evident (p=.09)

Table 2.

Adult ethanol consumption and BECs on 10th access session (30min). Means presented as mean (± SEM).

Sex Exposure Consumption (g/kg) BEC (mg/dl)
Female ethanol 1.27 (± 0.11) 6.47 (± 3.24)
water 1.58 (± 0.11) 20.26 (± 6.41)
Male ethanol 0.97 (± 0.11) 14.93 (± 7.45)
water 1.05 (± 0.15) 23.94 (± 8.78)
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Ethanol consumption (g/kg) and BECs as a function of Sex (male,female) and Exposure (ethanol,water).

Discussion

In the present study, intragastric administration of a moderate dose of ethanol (20% v/v; 2g/kg) during late gestation (G17–20) produced an attenuated sensitivity to ethanol-induced CTA in male adolescents when SS was used as the CS, but not when a weaker CS, NaCl, was used as the CS. Males that were prenatally exposed to ethanol also showed a notably greater preference for 5% ethanol relative to prenatally water-exposed animals when tested in young adulthood, an effect that emerged only gradually and was not evident in intake tests conducted during adolescence. In contrast to the significant impact of prenatal ethanol on ethanol CTA and intake observed in males, less notable effects were evident in females, with only modest evidence for a reduced preference for the 5% ethanol solution found among ethanol-exposed females.

These CTA findings are generally in agreement with previous studies that have found an attenuated CTA response to an ethanol or lithium chloride (LiCl) US following prenatal ethanol exposure using different species, rat strains and exposure periods. For example, Opitz et al. (1997) found that adult male mice (P54–59) that had received twice daily exposures of 1.58 g/kg ethanol from G14–18 did not show CTA to a 0.15% saccharin solution after it was paired with an intubation of 0.15M LiCl, while untreated control animals did. Likewise, previous work by Fabio et al. (2015) using the same dose and prenatal exposure as in the present study found that P44 male Wistars prenatally exposed to ethanol did not show CTA following a single 2.5g/kg i.p. injection of ethanol using NaCl as the CS, whereas untreated and prenatal water treated animals did.

The reason(s) why a prenatal exposure effect was found in males given SS but not NaCl is not known. There are a number of possibilities as to why the CTA response may differ with the CS used. One is that the tastants differed in the strength of their association with the aversive effects of ethanol. It appears that NaCl may have been a weaker CS than SS given that, for example, males did not develop a CTA at the lowest (1 g/kg) of ethanol when NaCl was used as the CS, whereas males given SS did demonstrate CTA by the third conditioning day at this dose. A less sensitive CS (NaCl) may have reduced between group differences in CTA and thus obscured detection of a prenatal exposure effect. Another possibility is that prenatal ethanol exposure enhanced the preference for SS and thereby affected the extent to which animals reduced their consumption of the CS over conditioning trials. Indeed, prenatal ethanol has been found to result in increased consumption of a novel saccharin solution (Gabriel & Weinberg, 2001) and to increase sucrose reinforcement due to common sensory and neurobiological substrates between ethanol and sweet tastants (Culleré et al., 2014). However, in the present study, differences in baseline SS intake were only seen in females, with both ethanol exposed and non-manipulated females consuming significantly more of the CS than water pre-exposed animals. Another important consideration is that there may be differences in CTA learning using a compound flavor (2 distinct flavors) vs a single flavor. Results from a study by Kucharski & Spear (1985) revealed that both preweanlings (P18) and adult (P60) Sprague-Dawley rats develop stronger aversions to compound flavors (ex: coffee/sucrose) compared to single flavors alone (ex: just sucrose). Therefore, the compound flavor of supersaccharin containing both sweet and bitter qualities may not produce a comparable CTA as when a single (salty) flavor is used.

Several studies have reported heightened ethanol intake in adolescents exposed to ethanol during late gestation (Chotro & Arias, 2003; Díaz-Cenzano & Chotro, 2010; Fabio et al., 2013; Fabio et al., 2015)., For instance, Fabio and colleagues (2015) used a 4-week intermittent access ethanol intake protocol similar to that of the present study (3 sessions/wk.;18 h per session) throughout most of adolescence (P37–62) and found significantly greater ethanol consumption and preference in animals prenatally exposed to ethanol than water (collapsed across sex); these effects on g/kg ethanol consumption and % preference for a 5% ethanol solution were seen on 8/12 and 6/12 intake sessions, respectively. In contrast, the present study did not find prenatal exposure differences on male ethanol preference from P34–55, although a notable increase in % preference and g/kg intake for a 5% ethanol solution was evident in testing occurring in older (P56–81) male offspring. One of the main differences between Fabio (2015) and the present procedure is the use of 2-bottle (vehicle, 5% ethanol) vs. 3-bottle (vehicle, 5% and 10% ethanol) tests, respectively. In the present study, both ethanol and water exposed adolescent males showed similar g/kg intake of the 10% and 5% ethanol solutions. It is possible that intake of the 10% ethanol solution may have limited intake of the 5% ethanol solution, thus obscuring exposure group differences that might have emerged if given the choice of only one ethanol solution and vehicle. This does not appear to be the case, however, for adult males prenatally exposed to ethanol, as they consumed roughly 3 times more of the 5% than the 10% ethanol solution in g/kg. Relative to water-exposed controls, the ethanol-exposed adult males’ significantly greater intake and preference for the 5% ethanol solution may have resulted in a corresponding decrease in their intake of the 10% ethanol solution. Note that this difference in intake of the 5% ethanol solution between ethanol- and water-exposed adult males was found in g/kg intake but not in percent preference. This finding suggests that prenatal ethanol exposure may result in offspring that, given the choice, preferentially consume solutions of lower ethanol concentration; in other words, that the effect of prenatal ethanol exposure on subsequent increased ethanol intake may be concentration-dependent. Future studies of prenatal ethanol exposure ought to consider using ethanol solutions of varying concentrations when assessing changes in voluntary consumption.

Reminiscent of the results of the present experiment, several studies using a range of fetal or neonatal ethanol exposure periods, typically including longer exposure periods, have also reported increased ethanol intake in adulthood (see Spear & Molina, 2005 for review). For example, Bond and DiGiusto (1977) reported greater consumption of ethanol by young adult offspring of dams that had been given a liquid diet consisting of 6.5% ethanol throughout gestation. This effect was evident only at low ethanol concentrations of 3–6%, findings reminiscent of the present study where differences in ethanol preference among adult males were only found at the 5%, but not the 10% ethanol concentration.

Unlike ethanol pre-exposed adult males, both adolescent and adult females that were pre-exposed to ethanol showed reduced preference and g/kg intake of a 5% ethanol solution compared to water exposed animals. This finding was unexpected given that Fabio et al. (2013) found no sex effects in their work, whereas Chotro & Arias (2003) found a male-specific increase using a 2 g/kg dose of ethanol and a female-specific increase using a 1 g/kg dose of ethanol during G17–20. While the reason for reduced preference and intake in the prenatal ethanol females relative to water exposed females is not known, it may be that the stress of intragastric intubations induced higher ethanol preference and consumption in this group, an effect that was reversed by potentially anxiolytic effects of prenatal ethanol. In support of this possibility, prenatal restraint stress has been shown to slightly increase ethanol preference in adolescent female, but not adolescent and adult male Sprague-Dawley rats (Van Waes et al., 2011a, 2011b). Interestingly, both adolescent and adult water exposed females had similar levels of preference (~50%) for the 5% ethanol solution as ethanol exposed adult males, while ethanol exposed females at both test ages had similar preference (~30%) and intake of the 5% ethanol solution as the water exposed adult males. This suggests that the stress of intragastric intubation in the water exposed adolescent and adult females may have increased preference and intake of the 5% ethanol solution, with prenatal ethanol exposure reversing this stress-induced effect.

One possible mechanism that could underlie increased ethanol preference after prenatal ethanol exposure is a reduced sensitivity to the aversive effects of ethanol. The results of the present study, however, do not provide evidence that attenuated CTA sensitivity is sufficient to increase ethanol consumption given the lack of a strong association between CTA and intake, with prenatal effects on CTA but not intake evident in adolescence whereas increased intake effects were only in adulthood. Given that CTA was not assessed in adult offspring, it is unclear whether these two variables would be associated in adulthood after prenatal ethanol exposure. While this study provides support for reduced sensitivity to aversive reinforcement in male offspring after prenatal ethanol exposure, we cannot rule out the possibility that this exposure might also have enhanced sensitivity to the appetitive effects of ethanol, thereby leading to escalated intake in adulthood. In support, the rat fetus during the G17–20 period has the capability to form associations between the orosensory characteristics of ethanol and its pharmacologically reinforcing properties (Chotro & Arias, 2006), which appear to be mediated by the opioid system (Chotro & Arias, 2003). Furthermore, infant rats prenatally exposed to 2 g/kg of ethanol from G17–20 have shown greater conditioned place preference during the ascending limb of the blood-ethanol curve (Pautassi et al., 2012) and more ingestive responses in reaction to the taste of ethanol compared to non-exposed animals (Arias & Chotro, 2005b). Part of this increased acceptability of ethanol following prenatal ethanol exposure may be taste-mediated. Work from Steve Youngentob’s group suggests that fetal ethanol exposure can enhance ethanol intake by attenuating sensitivity to the aversive bitter quinine-like qualities of ethanol while increasing response for the odor of ethanol (Youngentob & Glendinning, 2009).

Although we are unaware of any studies that have directly compared both sexes in response to ethanol-induced CTA in adolescence following prenatal ethanol exposure, there is evidence that CTA to ethanol and ethanol consumption may vary as a function of sex. For example, one study reported that in adults, but not adolescents, CTA to ethanol occurred at lower doses in females relative to males (Schramm-Sapyta et al., 2014) while another found that male adolescents were less sensitive to the aversive effects of ethanol than females when intoxication occurred in the presence of a peer (Vetter-O’Hagen et al., 2009).

Studies that have examined the effects of prenatal ethanol on later intake have often used a single sex or have not included sex as a variable (see Chotro et al., 2007 for review) making it difficult to draw definitive conclusions about the interaction of sex with prenatal ethanol exposure effects on voluntary ethanol intake. In the Chotro & Arias, (2003) article, however, sex differences in prenatal dose effects were observed, with elevated ethanol consumption evident in males only after prenatal exposure to 2 g/kg was given and in females only when 1 g/kg was given prenatally. This sex-specific effect on intake could be due to differential susceptibility to ethanol in females compared to males and/or sex-differences in pharmacokinetics (Robinson et al., 2002). Robinson and colleagues (2002) reported that following intragastric administration of 1 g/kg of ethanol, BECs peaked faster in females and reached the nucleus accumbens more quickly compared to males.

There are several limitations of the data that should be addressed. First, there was a Sex X Exposure difference in the number of low drinking animals that were excluded from the study for drinking less than 0.5g of the CS at baseline, specifically a greater number of ethanol exposed females were excluded compared to water and non-manipulated females, with no differences in the number of males removed. However, we do not believe this had a meaningful effect on our conclusions. In support, when all animals that were excluded from analysis are included all significant main effects, interactions and post-hocs are maintained for the male SS and NaCl CTA data. For females, the Exposure X Dose interaction in the SS CTA went from significant (p<.05) to marginally significant (p=.057), however the post-hocs on the data with low drinkers removed did not reveal any significant difference in consumption between exposure groups on the same day, therefore in both cases the conclusions have not changed. In addition, significant main effects, interactions and post-hocs for the NaCl CTA data remain unchanged in females.

Second, in experiment 1.1., females that were prenatally exposed to water consumed significantly less SS in g/kg at baseline compared to water exposed and non-manipulated females. To control for this we used a % saline analysis so that development of CTA was determined for each dose group relative to saline controls that had received the same prenatal exposure.

Third, there may be potential difficulties in the interpretation of % saline analysis from females in experiment 1.1 where there were differences in absolute baseline intake among exposure groups. However, since all groups consumed a sufficient amount as to avoid a floor effect, we do not believe that this would have had a large impact on the ability of water exposed females to develop a CTA. Moreover, the % saline analysis excluded baseline day, thereby mitigating the effect of different baseline intakes on % saline analysis. In addition, a repeated measures ANOVA comparing female SS g/kg and g consumption between all exposure groups (ethanol, water, non-manipulated) on days 2–4 revealed no differences. Therefore, the amount of consumption of saline groups likely had little bearing on the effect of CTA.

Fourth, among animals used in experiment 1.1 and 1.2, there were body weight differences with non-manipulated animals showing similar weights to ethanol-treated rather than water treated controls. We do not expect that body weight differences would have much of an effect on the interpretation of the results since development of CTA was determined within exposure groups. Findings from Uban et al., (2013) have found that the male and female offspring of dams that received an ethanol diet or pair-fed liquid control diet throughout gestation weighed less than control animals fed a standard lab chow at P1, but did not differ by P22. While these studies did not use intragastric intubation, our finding that the water intubated animals weighed more than ethanol and non-manipulated suggests that there may have been effect of intragastric intubation on weight that was reversed when combined with ethanol exposure.

Finally, it is worth noting that in all cases in experiment 2.1–2.2, differences in consumption of ethanol were only found with the 5% ethanol solution (except for a lower g/kg of the 10% ethanol solution in adult males).We believe that given the greater preference and intake (g/kg) of the 5% ethanol solution this may have obscured any differences in the preference and intake for the 10% ethanol solution if animals were only given this choice vs. vehicle.

In conclusion, a moderate dose of ethanol during late gestation was sufficient to attenuate sensitivity to the aversive effects of ethanol in adolescent males, but not females. Furthermore, this was accompanied by a male-specific increase in preference for a 5% ethanol solution during adulthood. Thus, while this study provides support that prenatal ethanol exposure may exacerbate adolescent typical insensitivity to ethanol, we could not confirm that this leads to heightened ethanol intake at this age. Future work is needed to disentangle the contribution of ethanol aversion or reward to normal age differences in ethanol intake, how that intake is perturbed by earlier ethanol exposure, and the neural substrates underlying these effects.

Supplementary Material

Supplemental 1

Highlights.

  • Prenatal ethanol reduced CTA response to ethanol in adolescent males

  • Prenatal ethanol did not affect ethanol intake in adolescent males

  • Increased preference and intake of 5% ethanol in adult males given ethanol prenatally

  • Reduced preference and intake of 5% ethanol in females given ethanol prenatally

Acknowledgments

Funding: This research was funded by the Developmental Exposure Alcohol Research Center (DEARC) P50 AA017823

Footnotes

Declarations of interest: none

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