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. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Alcohol Clin Exp Res (Hoboken). 2024 Feb 2;48(3):516–529. doi: 10.1111/acer.15267

A translational rodent model of individual differences in sensitivity to the aversive properties of ethanol

Kathryn R Przybysz 1, Lindsey A Ramirez 1, Joseph R Pitock 1, E Margaret Starr 1, Hyerim Yang 1, Elizabeth J Glover 1
PMCID: PMC10939790  NIHMSID: NIHMS1959735  PMID: 38303664

Abstract

Background.

A strong relationship exists between individual sensitivity to the aversive properties of ethanol and risk for alcohol use disorder (AUD). Despite this, our understanding of the neurobiological mechanisms underlying subjective response to ethanol is relatively poor. A major contributor to this is the absence of preclinical models that enable exploration of this individual variability similar to studies performed in humans.

Methods.

Adult male and female Long-Evans rats were trained to associate a novel tastant (saccharin) with acute exposure to either saline or ethanol (1.5 g/kg or 2.0 g/kg i.p.) over three conditioning days using a standard conditioned taste aversion (CTA) procedure. Variability in sensitivity to ethanol-induced CTA was phenotypically characterized using a median split across the populations studied.

Results.

When examining group averages, both male and female rats that had saccharin paired with either dose of ethanol exhibited reduced saccharin intake relative to saline controls of ethanol-induced CTA. Examination of individual data revealed a bimodal distribution of responses uncovering two distinct phenotypes present in both sexes. CTA-sensitive rats exhibited a rapid and progressive reduction in saccharin intake with each successive ethanol pairing. In contrast, saccharin intake was unchanged or maintained after an initial decrease from baseline levels in CTA-resistant rats. While CTA magnitude was similar between male and female CTA-sensitive rats, CTA-resistant females were more resistant to the development of ethanol-induced CTA than their male counterparts. Phenotypic differences were not driven by differences in baseline saccharin intake.

Conclusions.

These data parallel work in humans by revealing individual differences in sensitivity to the aversive properties of ethanol that emerge immediately after initial exposure to ethanol in both sexes. This model can be leveraged in future studies to investigate the neurobiological mechanisms that confer risk for AUD.

Keywords: subjective effects, phenotype, alcohol use disorder

Introduction

Heavy alcohol use can lead to a diagnosis of alcohol use disorder (AUD), which poses a significant health risk to affected individuals (Griswold et al., 2018; Key Substance Use and Mental Health Indicators in the United States: Results from the 2020 National Survey on Drug Use and Health, 2020). While much of the research aimed at understanding the neurobiological mechanisms underlying vulnerability for AUD has focused on the rewarding properties of ethanol, its aversive properties also affect the decision to drink (Riley, 2011; Verendeev & Riley, 2013). Indeed, while rewarding properties like euphoria, anxiolysis, and social facilitation, can promote continued drinking, ethanol’s aversive properties, which include sedation, motor impairment, and dysphoria, can limit drinking (A. C. King et al., 2019a; Riley, 2011; Verendeev & Riley, 2013). Importantly, individuals differ in their sensitivity to these properties, leading to individual variability in the subjective response to ethanol (Holdstock et al., 2000; A. C. King et al., 2002; Morean & Corbin, 2010). This subjective response is a significant risk factor for the development of AUD (Hines et al., 2005; Schuckit, 1999), such that individuals who are more sensitive to the rewarding properties of ethanol and less sensitive to its aversive properties are more likely to drink heavily (Holdstock et al., 2000; A. C. King et al., 2002, 2011; Verendeev & Riley, 2013). In fact, lower sensitivity to the aversive properties of ethanol is predictive of future binge drinking and AUD diagnosis (A. C. King et al., 2011, 2014).

Preclinical models using either ethanol-induced conditioned taste aversion (CTA) or conditioned place aversion (CPA) to assess response to the aversive properties of ethanol have successfully recapitulated the aforementioned clinical findings. For example, rat strains selectively bred for high ethanol drinking or preference exhibit less ethanol-induced CTA compared to their low drinking/preferring counterparts or their founder strains (Barkley-Levenson et al., 2015; Brunetti et al., 2002; Crabbe et al., 2019; Dyr et al., 2016; Wyszogrodzka et al., 2021). Similarly, DBA mice are more sensitive to ethanol-induced CTA than C57 mice, which typically drink higher quantities of ethanol than the DBA strain (Risinger & Cunningham, 1992). Studies assessing aversion to ethanol across multiple inbred mouse strains have also shown that stronger ethanol-induced CTA (Broadbent et al., 2002; Phillips et al., 2005) or CPA (Cunningham, 2019) was significantly correlated with lower ethanol intake or preference. Furthermore, a meta-analysis summarizing findings across 182 studies found that mouse strains with greater home-cage, voluntary ethanol intake exhibited lower ethanol-induced CTA or CPA (Green & Grahame, 2008). A more recent review drew similar conclusions, lending further support to the negative relationship between ethanol-induced CTA/CPA and drinking (Seemiller et al., 2022).

In addition to findings from inbred rodent strains, a number of studies have also shown differences in sensitivity to the aversive properties of ethanol across sexes and throughout development. For example, adolescent rodents are less sensitive to ethanol-induced CTA than adults, which likely contributes to greater levels of binge drinking in this population (Saalfield & Spear, 2016; Schramm-Sapyta et al., 2010, 2014; Vetter-O’Hagen et al., 2009a). Similarly, female rats, which typically consume greater quantities of ethanol than males, are less sensitive to ethanol’s aversive properties than males (Cailhol & Mormède, 2002; Roma et al., 2006, 2007; Schramm-Sapyta et al., 2014; Vetter-O’Hagen et al., 2009a) although this appears to vary by age (Schramm-Sapyta et al., 2014; Vetter-O’Hagen et al., 2009a) and strain (Cailhol & Mormède, 2002).

While these studies provide insight into the potential genetic and demographic factors contributing to risk for AUD, to our knowledge, no studies have examined individual differences in sensitivity to the aversive properties of ethanol in outbred animals of the same age or sex. Such comparisons are likely to have greater translational relevance to assessments done in the clinical population. To address this gap, the present study examined individual differences in ethanol-induced CTA in adult male and female outbred Long-Evans rats. Our results reveal that sensitivity to ethanol-induced CTA is bimodally distributed in both males and females, with some individuals exhibiting relatively high sensitivity and others relative resistance to CTA. The high degree of variability in sensitivity to the aversive properties of ethanol observed in this population parallels the individual differences in subjective response to ethanol observed in humans.

Materials & Methods

Animals

Adult male and female Long-Evans rats (Envigo, Indianapolis, IN) were P60 and weighed 336 ± 5.80 g and 200 ± 1.62 g, respectively, on arrival. Rats were allowed to acclimate to the vivarium for at least one week prior to experimental procedures. All rats were singly housed in standard cages in a temperature-controlled room on a reverse 12:12h light/dark cycle (lights on at 22:00). Food (Teklad 7912, Envigo) was provided ad libitum throughout the duration of the experiment. Water was available ad libitum prior to the onset of CTA procedures (see below). All procedures were approved by the University of Illinois Chicago Institutional Animal Care and Use Committee and adhered to the NIH Guidelines for the Care and Use of Laboratory Animals.

Conditioned taste aversion

Conditioned taste aversion was used to assess sensitivity to the aversive properties of ethanol using procedures described previously by our laboratory (Glover et al., 2016) (Figure 1A). In brief, rats were habituated to scheduled access to water for 30 min/day for seven days after which they were conditioned to associate a novel 0.1% saccharin solution with an intraperitoneal (i.p.) injection of either 1.5 g/kg ethanol (EtOH), 2.0 g/kg EtOH, or saline (EtOH-equivalent volume). Rats underwent three saccharin-drug conditioning days separated by 3–4 water recovery days. Bottle weights were taken before and after each session to measure fluid consumption during the 30-min access period. Rats receiving ethanol were assessed for behavioral signs of intoxication 30 min following injection using a previously published rating scale with scores ranging from 1 (no signs of intoxication) to 5 (loss of consciousness) (Glover et al., 2019, 2021; Trantham-Davidson et al., 2014). This time point was based on previous work by our laboratory showing that adult Long-Evans rats reach peak intoxication within 30 minutes of i.p. ethanol administration (Glover et al., 2021). Of note, while drug injection is typically omitted after the final saccharin exposure session, rats in this experiment continued saccharin-drug conditioning beyond the time frame reported in the current manuscript in order to address a separate research question (reported in Ramirez et al., 2023). Importantly, the data presented in the current manuscript is limited to saccharin intake on conditioning days 1–3 and therefore is unaffected by drug injection after saccharin consumption on day 3.

Figure 1. Dose-dependent increase in ethanol-induced CTA in adult male and female Long-Evans rats.

Figure 1.

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(A) Rats underwent three conditioning sessions during which 0.1% saccharin was followed by injection of either saline, 1.5 g/kg EtOH, or 2.0 g/kg EtOH. Each conditioning day was separated by three water recovery days. (B) Change in saccharin intake in males and females normalized to baseline as percent change revealed a significant difference in intake between saline and 1.5 g/kg and 2.0 g/kg EtOH groups on conditioning days 2 and 3. Intake was also significantly different between the 1.5 g/kg and 2.0 g/kg EtOH groups on conditioning day 3. Significant changes in intake from baseline were also observed on conditioning days 2 and 3 for rats within each sex/drug group. Both male (C) and female (D) rats injected with 1.5 g/kg or 2.0 g/kg EtOH drank significantly less saccharin than saline-injected rats on conditioning days 2 and 3. Rats injected with 2.0 g/kg EtOH also drank significantly less saccharin than rats injected with 1.5 g/kg EtOH on conditioning day 3. *p<0.0001 saline compared to both EtOH doses; §p≤0.05 compared to 1.5 g/kg EtOH; ^p<0.05 compared to conditioning day 1.

Phenotypic classification

Rats were categorized into CTA-sensitive and -resistant phenotypes upon completion of the study using a median split of the percent change in saccharin intake from baseline (conditioning day 1, prior to drug injection) to conditioning day 3. Rats with a percent change in intake that was numerically below the median, indicating greater magnitude CTA, were classified as CTA sensitive. Conversely, rats with a percent change in intake that was numerically above the median, indicating lower magnitude CTA, were classified as CTA resistant.

Blood ethanol concentration during CTA

A separate cohort of rats from those described above were used to determine the relationship between blood ethanol concentration (BEC) and CTA magnitude. CTA was performed as described above with saccharin paired with either 1.5 g/kg i.p. EtOH or saline. Blood samples were obtained from ethanol-injected rats 15 min following injection via tail vein nick. Immediately following collection, blood samples were centrifuged (10,000 x g, 10 min, 4°C). Plasma supernatant (20 μL) was aliquoted into 0.5 mL microcentrifuge tubes and stored at −20°C until ready for processing. BECs were measured using an Analox Alcohol Analyzer (Analox Instruments Ltd, United Kingdom) within 30 days of sample collection.

Statistical analysis

CTA was measured by saccharin intake (mL) uncorrected for body weight. This approach is based on data showing that the relationship between fluid intake and body weight is non-linear and that weight-corrected data is not optimal for comparing intake across sexes (Richter & Brailey, 1929; Holdstock, 1973; Santollo & Edwards, 2021). However, because a threshold level effect was observed in fluid intake between male and female rats, comparisons made across sexes were performed using percent change in saccharin intake as the dependent measure. Comparisons between phenotypes were analyzed using multifactorial analysis of variance (ANOVA), unpaired t-tests, and one-sample t-tests as described below. Spearman correlations were used to assess the relationship between intoxication score and percent change from baseline saccharin intake. Linear regressions were used to assess the relationship between BEC and percent change from baseline saccharin intake. Where appropriate, post-hoc analyses were performed using Tukey’s HSD test. ROUT outlier tests were run on all parameters. The initial sample size for the main CTA experiment was n=26/sex/group. However, one female in the 1.5 g/kg EtOH group was removed from the study due to excessive weight loss. Another female from the 1.5 g/kg EtOH group and one female from the 2.0 g/kg EtOH group were identified as statistical outliers and removed from all subsequent analyses. Data from one additional female in the saline group was removed due to a technical error resulting in inaccurate measurement of fluid consumption. This resulted in the following final sample sizes: female saline n=25; female 1.5 g/kg EtOH n=24; female 2.0 g/kg EtOH n=25, and n=26 in all three drug groups in males. No subjects were excluded from the cohort used to assess the relationship between CTA magnitude and BEC, which included rats that had saccharin paired with saline (n=3/sex) or 1.5 g/kg EtOH (n=12/sex). All statistical tests and graphs were generated using Prism 9 (Graphpad Software, San Diego, CA) or SPSS (IBM, New York). Data are reported as mean ± SEM.

Results

Ethanol induces significant conditioned taste aversion in males and females

To determine the extent of ethanol-induced CTA at each of the two doses administered, we conducted an omnibus ANOVA on saccharin intake with conditioning day, drug group, and sex as factors. This analysis uncovered a significant main effect of sex [F(1, 146)=70.650, p<0.0001], which showed that, overall, males consumed significantly more saccharin than females independent of drug group. Therefore, to better determine whether sex differences in CTA magnitude were present, raw intake data were normalized to percent change in intake from baseline (Figure 1B). Using this approach, there were significant main effects of conditioning day [F(1, 146)=26.31, p<0.001] and drug [F(2, 146)=85.61, p<0.001], but not sex [p>0.05]. There was also a significant conditioning day x drug interaction [F(4, 292)=56.91, p<0.001]. Post-hoc analyses revealed a significant difference between saline and 1.5 g/kg EtOH on conditioning days 2 [p<0.001] and 3 [p<0.001], as well as a significant difference between saline and 2.0 g/kg EtOH on conditioning days 2 [p<0.001] and 3 [p<0.001]. Additionally, a significant difference between 1.5 g/kg EtOH and 2.0 g/kg EtOH was present on conditioning day 3 [p=0.014]. To more directly assess changes in saccharin intake from baseline across conditioning days within each drug group and sex, we also performed one-sample t-tests on conditioning days 2 and 3 in these groups. In males on conditioning day 2, there was a significant increase from baseline in the saline group [t(25)=7.11, p<0.001] and a significant decrease from baseline in the 1.5 g/kg EtOH group [t(25)=3.83, p=0.0008] and the 2.0 g/kg EtOH group [t(25)=6.59, p<0.0001]. Similarly, on conditioning day 3, there was also a significant increase from baseline in the saline group [t(25)=7.34, p<0.0001] and a significant decrease from baseline in the 1.5 g/kg EtOH group [t[25)=6.07, p<0.0001] and the 2.0 g/kg EtOH group [t(25)=7.33, p<0.0001]. In females on conditioning day 2, we found a significant increase from baseline in the saline group [t(24)=4.26, p=0.0003], a trend toward a decrease from baseline in the 1.5 g/kg EtOH group [t(23)=1.86, p=0.07], and a significant decrease from baseline in the 2.0 g/kg EtOH group [t(24)=2.69, p=0.01]. On conditioning day 3 in females, we again found a significant increase from baseline in the saline group [t(24)=4.18, p=0.0003], as well as a significant decrease from baseline in both the 1.5 g/kg EtOH group [t(23)=3.39, p=0.0025] and the 2.0 g/kg EtOH group [t(24)=7.59, p<0.0001].

Similar results were obtained using a two-way repeated measures mixed ANOVA with conditioning day and drug as factors to analyze changes in raw intake separately for males and females. Thus, in males, this analysis revealed a significant main effect of conditioning day [F(1.86, 139.5)=17.86, p<0.0001], a significant main effect of drug [F(2, 75)=47.29, p<0.0001], and a significant conditioning day x drug interaction [F(4, 150)=44.55, p<0.0001]. Post-hoc analyses showed significantly lower saccharin intake in the 1.5 g/kg EtOH group compared to saline on conditioning days 2 [q(41.42)=10.53, p<0.0001] and 3 [q(36.55)=12.26, p<0.0001]. This was also true for the 2.0 g/kg EtOH group relative to saline controls at the same time points [day 2: q(46.80)=15.97, p<0.0001; day 3: q(41.36]=18.94, p<0.0001]. Saccharin intake was also significantly lower on conditioning day 3 in the 2.0 g/kg EtOH group compared to intake in the 1.5 g/kg EtOH group on the same day [q(48.04)=3.397, p≤0.05] (Figure 1C).

In females, analysis of saccharin intake across days revealed significant main effects of conditioning day [F(1.876, 133.2)=10.51, p<0.0001], and drug [F(2, 71)=34.18, p<0.0001], as well as a significant conditioning day x drug interaction [F(4, 142)=20.78, p<0.0001]. Post-hoc analyses revealed findings similar to those observed in the males. Specifically, rats that had 1.5 g/kg EtOH paired with saccharin had significantly lower saccharin intake compared to saline controls on conditioning days 2 [q(38.48)=6.761, p<0.0001] and 3 [q(45, 49)=9.462, p<0.0001]. Similarly, the 2.0 g/kg EtOH group exhibited significantly lower saccharin intake compared to saline on both conditioning days [day 2: q(43.21)=9.664, p<0.0001; day 3: q(47.46)=14.46, p<0.0001]. The 2.0 g/kg EtOH group also had significantly lower saccharin intake compared to the 1.5 g/kg EtOH group on conditional day 3 [q(46.73)=4.339, p=0.001] (Figure 1D). Taken together, these data demonstrate the development of dose-dependent ethanol-induced CTA of similar magnitude between male and female rats.

Individual differences in sensitivity to ethanol-induced CTA

To determine whether individual differences in sensitivity to the aversive properties of ethanol could be categorized into distinct phenotypes, we calculated the percent change in saccharin intake from baseline, prior to any ethanol exposure, to intake on conditioning day 3, after two saccharin-ethanol pairings. This analysis revealed a bimodal distribution of responses present in both males (Figure 2A,2D) and females (Figure 3A,3D) at both doses tested. For one cluster of rats, the percent change in saccharin intake was relatively close to zero, indicating minimal reduction in consumption across conditioning days. In the other cluster of rats, the percent change in saccharin intake was substantially below zero, indicating a large reduction in consumption across conditioning days. We refer to these clusters as CTA-resistant and -sensitive, respectively. Importantly, saccharin intake did not differ significantly at baseline between phenotypes. This was evident in male rats injected with 1.5 g/kg EtOH using a t-test comparing saccharin intake on conditioning day 1 between rats classified as CTA resistant and CTA sensitive [t(16.46)=1.95, p>0.05; with Welch’s correction due to significant between-group differences in variance] (Figure 2B). Likewise, no difference in baseline saccharin intake was observed between phenotypes in males injected with 2.0 g/kg EtOH [t(24)=0.467, p>0.05] (Figure 2E). Similar to males, we observed no significant difference in baseline saccharin intake between phenotypes in females at either dose [1.5 g/kg EtOH: t(22)=1.282, p>0.05; 2.0 g/kg EtOH: t(23)=0.205, p>0.05] (Figures 3B, 3E). In addition, linear regression found no significant relationship between baseline saccharin intake and percent change in saccharin intake in males at either dose [1.5 g/kg: r2=0.097, p>.0.05; 2.0 g/kg: r2=0.123, p>0.05 (not shown)]. A similar absence of significance was observed when the same analysis was performed in females [1.5 g/kg: r2=0.046, p>0.05; 2.0 g/kg: r2=0.025, p>0.05 (not shown)]. Together, these data suggest that sensitivity to ethanol-induced CTA is not driven by innate differences in hedonic drive for saccharin in either males or females.

Figure 2. Individual differences in sensitivity to ethanol-induced CTA in male Long-Evans rats.

Figure 2.

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A bimodal distribution in sensitivity to ethanol-induced CTA is apparent in rats injected with either 1.5 g/kg (A) or 2.0 g/kg (D) EtOH when individual data is plotted as percent change from baseline in saccharin intake on conditioning day 3. Rats were classified as CTA sensitive or CTA resistant using a median split. Baseline saccharin intake (prior to pairing with EtOH) was not significantly different between CTA-resistant and -sensitive rats in either the 1.5 g/kg EtOH group (B) or the 2.0 g/kg EtOH group (E). Average saccharin intake differed between phenotypes across conditioning days. In both the 1.5 g/kg EtOH group (C) and the 2.0 g/kg EtOH group (F), both CTA-resistant and -sensitive rats drank significantly less saccharin than saline controls on conditioning days 2 and 3. CTA-sensitive rats in both drug groups also drank significantly less saccharin than CTA-resistant rats on conditioning days 2 and 3. *p<0.002 compared to saline; +p<0.01 compared to CTA-resistant. In A and D, dashed lines indicate the median and dotted lines indicate the upper and lower quartiles.

Figure 3. Individual differences in sensitivity to ethanol-induced CTA in female Long-Evans rats.

Figure 3.

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Similar to males, a bimodal distribution in sensitivity to ethanol-induced CTA is apparent in rats injected with either 1.5 g/kg (A) or 2.0 g/kg (D) EtOH when individual data is plotted as percent change from baseline in saccharin intake on conditioning day 3. Rats were classified as CTA-sensitive or CTA-resistant using a median split. Baseline saccharin intake (prior to pairing with EtOH) was not significantly different between CTA-resistant and -sensitive rats in either the 1.5 g/kg EtOH group (B) or the 2.0 g/kg EtOH group (E). Average saccharin intake differed between phenotypes across conditioning days. In both the 1.5 g/kg EtOH group (C) and the 2.0 g/kg EtOH group (F), both CTA-resistant and -sensitive rats drank significantly less saccharin than saline controls on conditioning days 2 and 3. CTA-sensitive rats injected with 1.5 g/kg EtOH (C) also drank significantly less saccharin than CTA-resistant rats on conditioning day 3. In contrast, the difference in intake between CTA-sensitive and -resistant phenotypes injected with 2.0 g/kg EtOH (F) was significant on conditioning days 2 and 3. *p<0.002 compared to saline; +p<0.01 compared to CTA-resistant. In A and D, dashed lines indicate the median and dotted lines indicate the upper and lower quartiles.

To determine whether the emergence of ethanol-induced CTA was significantly different between phenotypes, we next analyzed saccharin intake across conditioning days by comparing rats that received EtOH to saline controls. In males that received saccharin paired with 1.5 g/kg EtOH, a two-way mixed ANOVA revealed significant main effects of conditioning day [F(1.90, 93.19=6.933, p=0.002], and of phenotype [F(2, 49)=49.47, p<0.0001] and a conditioning day x phenotype interaction [F(4, 98= 43.9, p<0.0001] (Figure 2C). Post-hoc analyses showed that, while there were no baseline differences in saccharin intake between CTA-resistant or -sensitive rats compared to saline-injected rats, intake diverged significantly between groups on later conditioning days. Both CTA-sensitive and -resistant rats consumed significantly less saccharin compared to the saline group on conditioning days 2 [sensitive: q(21.6)=14.28, p<0.0001; resistant: q(36.49)=2.062, p=0.002] and 3 [sensitive: q(18.62)=17.97, p<0.0001; resistant: q(30.29)=10.61, p<0.0001]. CTA-sensitive rats also had significantly lower saccharin intake compared to CTA-resistant rats on conditioning days 2 [q(21.74)=4.74, p=0.008] and 3 [q(18.95)=10.54, p<0.0001].

Similar results were uncovered when comparing rats injected with 2.0 g/kg EtOH. A two-way mixed ANOVA found significant main effects of conditioning day [F(1.981, 97.08)=30.13, p<0.0001], and phenotype [F(2, 49)=93.26, p<0.0001], as well as a significant interaction between these two factors [F(4. 98)=70.87, p<0.0001] (Figure 2F). Post-hoc analyses found that both CTA-resistant and -sensitive rats consumed significantly less saccharin compared to saline-injected rats on conditioning days 2 [resistant: q(25.21)=12.71, p<0.0001; sensitive: q(19.6)=15.12, p<0.0001] and 3 [resistant: q(26.02)=15.75, p<0.0001; sensitive: q(34.97)=41.62, p<0.0001]. In addition, CTA-sensitive rats injected with 2.0 g/kg EtOH consumed significantly less saccharin than rats in the resistant phenotype on the second [q(22.19)=4.392, p=0.014] and third [q(15.71)=15.03, p<0.0001] conditioning days. Taken together, these data reveal two distinct phenotypes in male Long-Evans rats, with CTA-sensitive rats exhibiting significantly greater magnitude ethanol-induced CTA relative to CTA-resistant rats at each dose tested.

The same analysis in females produced results comparable to our observations in males. Specifically, in the 1.5 g/kg EtOH group, there was a significant main effect of phenotype [F(2, 47)=18.81, p<0.0001] and a conditioning day x phenotype interaction [F(4, 94)=19.30, p<0.0001], but no main effect of conditioning day (Figure 3C). Post-hoc analyses found significantly lower saccharin intake in both CTA-resistant [q(17.81)=5.154, p=0.005] and -sensitive phenotypes [q(16.02)=5.655, p=0.003] relative to saline on conditioning day 2. CTA-resistant females also consumed significantly less saccharin compared to saline-injected females on the third conditioning day [q(28.74)=5.562, p=0.001], as did CTA-sensitive females [q(30.47)=13.75, p<0.0001]. However, unlike in males injected with the same dose of ethanol, the difference in saccharin intake between CTA-sensitive and -resistant females did not emerge until the third conditioning day [q(22.97)=7.968, p<0.0001]. Comparisons between phenotypes in females injected with 2.0 g/kg EtOH uncovered significant main effects of conditioning day [(F(1.963, 94.20)=14.50, p<0.0001] and phenotype [F(2, 48)=49.24, p<0.0001] as well as a significant conditioning day x phenotype interaction [F(4, 96)=22.63, p<0.0001]. Post-hoc analyses found significantly lower saccharin intake in both phenotypes compared to saline controls on the second [resistant: q(17.79)=5.815, p=0.0018; sensitive: q(19.16)=10.49, p<0.0001] and third [resistant: q(33.84)=8.033, p<0.0001; sensitive: q(31.47)=19.51, p<0.0001] conditioning days. In contrast to females injected with 1.5 g/kg EtOH, saccharin intake was significantly lower in CTA-sensitive females injected with 2.0 g/kg EtOH compared to CTA-resistant females on both conditioning day 2 [q(22.98) = 3.82, p = 0.033] and 3 [q(15.31)=11.04, p<0.0001]. Taken together, these data indicate that, similar to males, female CTA-sensitive rats exhibit stronger ethanol-induced CTA than CTA-resistant rats. However, in contrast to males, the difference between CTA-sensitive and -resistant phenotypes in females injected with 1.5 g/kg EtOH was not evident until the third conditioning day.

Dose and sex differences in the emergence of ethanol-induced CTA across phenotypes

To more explicitly examine whether the rate at which CTA sensitivity emerged differed across phenotypes and ethanol dose, we assessed within-group differences in the percent change in saccharin intake from baseline on conditioning days 2 and 3. We hypothesized that ethanol-induced CTA would emerge after fewer pairings in the 2.0 g/kg EtOH dose compared to the 1.5 g/kg EtOH dose in both male and female rats. In rats injected with 1.5 g/kg EtOH, one-sample t-tests on conditioning day 2 revealed that saccharin intake was unchanged in CTA-resistant male and female rats (p>0.05). In contrast, CTA-sensitive rats exhibited a significant decrease in saccharin intake from baseline ([males: t(12)=4.888, p=0.0004; females t(12)=2.736, p=0.018] (Figure 4A). As expected, CTA-sensitive male and female rats continued to exhibit a reduce saccharin intake from baseline on the third conditioning day [males: t(12)=10.55, p<0.0001; females: t(12)=10.49, p<0.0001]. Interestingly, by the third conditioning day, CTA-resistant males exhibited a significant reduction in intake [t(12)=3.676, p=0.003], however, intake in CTA-resistant females still did not differ significantly from baseline levels [p>0.05] (Figure 4B). CTA-sensitive rats of both sexes injected with 2.0 g/kg EtOH exhibited a significant reduction in saccharin intake from baseline on conditioning day 2 [males: t(12)=7.2, p<0.0001; females: t(12)=3.274, p=0.007]. One-sample t tests also showed that CTA-resistant males had significantly reduced saccharin intake on conditioning day 2 after 2.0 g/kg EtOH [t(12)=3.671, p=0.003]. In contrast, CTA-resistant females injected with 2.0 g/kg EtOH did not reduce their saccharin intake on the second conditioning day [p>0.05] (Figure 4C). By the third conditioning day, CTA-resistant and -sensitive rats of both sexes that had saccharin paired with 2.0 g/kg EtOH exhibited a significant reduction in saccharin intake relative to baseline [CTA-resistant males: t(12)=2.839, p=0.01; CTA-resistant females: t(11)=3.166, p=0.009; CTA-sensitive males: t(12)=47.06, p<0.0001; CTA-sensitive females: [t(12)=25.98, p<0.0001] (Figure 4D). Altogether, these data uncover significant sex differences within the CTA-resistant phenotype with CTA-resistant females exhibiting greater resistance to ethanol-induced changes in saccharin intake than CTA-resistant males. This difference is eliminated after multiple pairings with high dose ethanol (2.0 g/kg) indicative of a dose-dependent increase in ethanol-induced CTA independent of phenotype.

Figure 4. Phenotype-specific sex differences in CTA magnitude.

Figure 4.

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(A) One-sample t-tests comparing percent change in saccharin intake from baseline to conditioning day 2 in rats of each sex revealed a significant decrease in intake on conditioning day 2 in CTA-sensitive male (M) and female (F) rats injected with 1.5 g/kg EtOH. In contrast, no significant change from baseline was observed in CTA-resistant rats of either sex. (B) By the third conditioning day, male CTA-resistant rats injected with 1.5 g/kg EtOH exhibited a significant reduction in saccharin intake from baseline levels, whereas saccharin intake in female CTA-resistant rats remained unchanged from baseline. (C) In the 2.0 g/kg EtOH group, CTA-resistant males exhibited a significant reduction in saccharin intake on conditioning day 2, whereas CTA-resistant females did not. (D) By the third conditioning day, both male and female CTA-resistant rats had significantly decreased their saccharin consumption relative to baseline levels, albeit to a lesser degree than CTA-sensitive rats. In contrast, CTA-sensitive rats of both sexes exhibited a significant decrease in saccharin intake on both conditioning days. Significance indicators reflect comparisons to baseline not across sexes: *p<0.05, **p<0.01, ***p<0.001, ns=not significant.

Relationship between intoxication and CTA phenotype

To determine whether behavioral signs of intoxication were distinct between CTA phenotypes, we examined intoxication scores across sex, ethanol dose, phenotype, and conditioning days. A multifactorial ANOVA revealed an expected main effect of ethanol dose [F(1, 79)=13.19, p<0.001], but no main effect of conditioning day, sex, or phenotype (all p>0.05). Based on these results, we next performed separate intoxication score analyses for each ethanol dose group while collapsing these data across sex (Figures 5 and 6).

Figure 5. Relationship between behavioral signs of intoxication and CTA phenotype in male and female rats injected with 1.5 g/kg EtOH.

Figure 5.

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(A) CTA-resistant and -sensitive male and female rats exhibited similar levels of intoxication when measured as average intoxication score across conditioning days. (B) Intoxication scores were also similar between phenotypes when assessed on individual conditioning days. Black lines and data points represent data averaged across sexes. Data by sex is shown in colored lines for transparency purposes. (C) CTA magnitude was not significantly correlated with intoxication score on conditioning day 1 or (D) the average intoxication score achieved across conditioning days.

Figure 6. Relationship between behavioral signs of intoxication and CTA phenotype in male and female rats injected with 2.0 g/kg EtOH.

Figure 6.

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(A) CTA-resistant and -sensitive male and female rats exhibited similar levels of intoxication when measured as average intoxication score across conditioning days. (B) Intoxication scores were also similar between phenotypes when assessed on individual conditioning days. Black lines and data points represent data averaged across sexes. Data by sex is shown in colored lines for transparency purposes. CTA magnitude was significantly positively correlated with intoxication score on conditioning day 1 (C) but not when comparisons were made with (D) average intoxication score achieved across conditioning days.

We first compared average intoxication scores between phenotypes in male and female rats that received saccharin paired with 1.5 g/kg EtOH. This analysis showed no difference between phenotypes (p>0.05) (Figure 5A). To examine this more closely, we considered whether intoxication differed significantly between phenotypes during distinct phases of the CTA paradigm by comparing intoxication scores across conditioning days. A two-way ANOVA with phenotype and conditioning day as factors also found no significant effects of conditioning day or phenotype and no day x phenotype interaction (p>0.05) (Figure 5B). Given the possibility that level of intoxication may be one explanation for apparent differences in CTA magnitude between phenotypes, we also performed a Spearman correlation between intoxication score on each conditioning day and percent change in saccharin intake. Using this approach, we found no significant relationship between CTA magnitude and intoxication score on conditioning day 1 (r=0.045; p=0.756) (Figure 5C) or the average intoxication score achieved across conditioning days 1 and 2 (r=0.109; p=0.454) (Figure 5D).

Similar to the 1.5 g/kg EtOH group, there was no significant difference in average intoxication score between phenotypes in rats injected with 2.0 g/kg EtOH (p>0.05) (Figure 6A). Likewise, a two-way ANOVA found no significant main effects or interaction between conditioning day and phenotype (p>0.05) (Figure 6B). Interestingly, a Spearman correlation revealed a significant negative relationship (r=−0.30; p=0.041) between intoxication score on conditioning day 1 and percent change in saccharin intake in rats injected with 2.0 g/kg EtOH (Figure 6C). However, this relationship did not remain significant when the same analysis was performed with the average intoxication score across conditioning days 1 and 2 (r=−0.06; p-0,712) (Figure 6D). Taken together, these data suggest that while degree of intoxication experienced during conditioning – as measured by behavioral signs – is not a primary driver of CTA magnitude, at least in the majority of individuals, it may be influential during the first exposure to ethanol at higher doses (e.g., 2.0 g/kg).

While measuring the behavioral signs of intoxication via intoxication score is a non-invasive way to assess overall levels of intoxication, the narrow range of possible scores (nearly all rats achieved a score of 2 or 3) limits the capacity to uncover meaningful correlations between intoxication and CTA magnitude. To address this limitation, we measured BECs achieved after each injection of ethanol in a separate cohort of rats that underwent the same CTA paradigm with saccharin paired with either saline or 1.5 g/kg EtOH (Figure 7). Rats were categorized as CTA-sensitive or -resistant using the same median split approach as used above. Similar to the results shown in Figures 1–3, a multifactorial ANOVA including conditioning day, sex, drug injected, and phenotype on saccharin intake revealed a significant main effect of sex [F(1, 24)=7.62, p=0.011], with males again consuming significantly more saccharin than females. Data from males and females were therefore separated in all subsequent analyses of saccharin intake. When comparing saccharin intake in male rats injected with 1.5 g/kg EtOH to saline controls across conditioning days, we found a significant main effect of conditioning day [F(1.54, 18.43)=3.853, p=0.049] and of phenotype [F(2, 12)=9.316, p=0.003], as well as a day x phenotype interaction [F(4, 24)=9.929, p<0.0001] (Figure 7A). Post-hoc analysis showed that, while there were no significant differences in saccharin intake at baseline (p>0.05 for all comparisons), saccharin intake was significantly lower in male rats that received 1.5 g/kg EtOH on subsequent conditioning days compared to saline-injected rats. In addition, the level of intake differed between phenotypes. Specifically, on conditioning day 2, CTA-sensitive males consumed significantly less saccharin compared to saline controls (q(5.16)=4.85, p=0.039), whereas CTA-resistant rats did not differ from controls (q(5.79)=2.02, p>0.05). On conditioning day 3, however, both CTA-sensitive and -resistant males consumed significantly less saccharin compared to saline controls [CTA sensitive: q(6.125)=14.74, p<0.0001; CTA resistant: q(6.99)=6.50, p=0.006]. CTA-sensitive males also drank significantly less saccharin compared to CTA-resistant males on conditioning day 3 [q(8.72)=4.75, p=0.021]. The same analyses produced similar results in females (Figure 7B). Thus, a significant main effect of conditioning day [F(1.492, 17.91)=11.40, p=0.001] and a significant day x phenotype interaction [F(4, 24)=6.941, p=0.0007] were observed in females in the absence of a main effect of phenotype [F(2, 12)=2.183, p>0.05). Post-hoc analyses showed that there was no difference in baseline saccharin intake, nor were there between-group differences on conditioning day 2 (p>0.05 for all comparisons). However, on conditioning day 3, CTA-sensitive females consumed significantly less saccharin intake compared to saline controls [q(3.47)=6.38, p=0.03] and compared to CTA-resistant females [q(9.48)=3.79, p=0.05]. Altogether, these data replicate our previous findings in a separate cohort of rats and confirm the presence of two distinct phenotypes exhibiting differential sensitivity to ethanol-induced CTA.

Figure 7. Relationship between BEC and CTA phenotype in male and female rats injected with 1.5 g/kg EtOH.

Figure 7.

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The same CTA-sensitive and -resistant phenotypes were observed in a separate cohort of male (A) and female (B) rats with greater reduction in saccharin intake observed in CTA-sensitive than -resistant rats of both sexes. (C) BECs achieved 15 min after EtOH injection did not differ between phenotypes across conditioning days. Black lines and data points represent data averaged across sexes. Data by sex is shown in colored lines for transparency purposes. CTA magnitude was not significantly correlated with BEC on the first conditioning day (D) or average BEC (E) achieved across conditioning trials. *p≤0.05 compared to saline controls; +p≤0.05 compared to CTA-resistant group.

Next, we used a multifactorial ANOVA to examine the relationship between BEC and CTA magnitude with sex, phenotype, and conditioning day as factors. This analysis produced no significant main effects or interactions (p>0.05 for all comparisons) (Figure 7C), indicating that BEC did not differ between groups or across conditioning days. Linear regression was used to investigate this more closely, however, no significant correlation was observed between BECs achieved after ethanol injection on conditioning day 1 or average BECs across conditioning days and percent change in saccharin intake [conditioning day 1: r2=0.001, p>0.05 (Figure 7D); average BEC: r2=0.094, p>0.05 (Figure 7F). These data lend support to the idea that degree of intoxication is not the primary driver of differences in CTA sensitivity.

Discussion

Clinical studies have shown that individuals who are more responsive to the rewarding properties of ethanol and less responsive to its aversive properties are at higher risk for future binge drinking and AUD diagnosis (A. King et al., 2021; A. C. King et al., 2002, 2011, 2014, 2019b). This relationship has been consistently and extensively recapitulated in rodent models (e.g., Barkley-Levenson et al., 2015; Brunetti et al., 2002; Crabbe et al., 2019; Dyr et al., 2016; Wyszogrodzka et al., 2021) including in a meta-analysis of data from 182 different studies (Green & Grahame, 2008; see Seemiller et al., 2022 for review). However, preclinical findings have been limited to genetically and/or demographically distinct subpopulations (e.g., alcohol-preferring vs non-preferring; adult vs adolescent), unlike studies performed in clinical populations. Using CTA, results from the present study uncovered significant individual differences in response to the aversive properties of ethanol in adult male and female outbred Long-Evans rats. As expected, when examining group averages, the magnitude of ethanol-induced CTA was dose-dependent. However, closer inspection of individual responses revealed a bimodal distribution in CTA expression in both sexes at both doses. Thus, some individuals displayed a CTA-sensitive phenotype and others appeared relatively resistant to the development of ethanol-induced CTA. While the magnitude of ethanol-induced CTA was similar between male and female rats classified as CTA-sensitive, we observed noteworthy differences in the emergence of CTA in male and female CTA-resistant rats, with CTA-resistant females requiring more conditioning trials to develop significant ethanol-induced CTA than CTA-resistant males. Altogether, these data uncover important individual differences in ethanol-induced CTA in a rodent model that more closely reproduces the variability observed in clinical samples than has been reported in previous preclinical studies.

We observed significant dose-dependent ethanol-induced CTA in adult male and female Long Evans rats, with 2.0 g/kg EtOH producing greater CTA magnitude than 1.5 g/kg EtOH in both sexes. These data are in agreement with prior research showing that ethanol becomes more aversive as dose increases (Cunningham, 2019; Moore et al., 2013; Morales et al., 2014; Phillips et al., 2005; Saalfield & Spear, 2016; Schramm-Sapyta et al., 2014; Vetter-O’Hagen et al., 2009b). Analysis of the distribution of CTA magnitude across individuals uncovered two distinct phenotypes present in both male and female rats: a CTA-sensitive group that exhibited the expected reduction in saccharin intake as the number of saccharin-ethanol pairings increased, and a CTA-resistant group that exhibited little-to-no reduction in saccharin intake across conditioning days. While both CTA-sensitive and -resistant phenotypes drank significantly less saccharin than saline controls over the course of conditioning, CTA-sensitive rats exhibited a rapid decrease in saccharin intake, reducing their intake by almost half after just one saccharin-ethanol pairing. This was followed by an even greater reduction in intake after the second saccharin-ethanol pairing. In contrast, CTA-resistant rats exhibited minimal reduction in saccharin intake after one saccharin-ethanol pairing. The majority of CTA-resistant rats then maintained this level of intake despite an additional conditioning day.

Notably, unlike males, CTA-resistant females that had saccharin paired with 1.5 g/kg ethanol exhibited a slight increase in saccharin intake over conditioning days, suggesting a near complete lack of sensitivity to the aversive properties of ethanol. This observation led us to more closely examine the rate at which rats within each phenotype developed CTA by assessing the percent change in saccharin intake from baseline on each conditioning day. These comparisons revealed that both male and female CTA-resistant rats maintained baseline levels of saccharin intake after a single pairing with 1.5 g/kg ethanol. While males reduced their intake significantly from baseline, albeit only slightly, female rats continued to maintain baseline levels of intake at this dose after an additional saccharin-ethanol pairing. Similar findings were observed in females after a single pairing with 2.0 g/kg ethanol, although this dose was sufficient to produce a slight, but significant, reduction in intake after two saccharin-ethanol pairings. In contrast, all CTA-sensitive rats exhibited a significant decrease in saccharin intake after a single saccharin-ethanol pairing regardless of sex or ethanol dose. Thus, our data indicate that sex differences in ethanol-induced CTA are driven primarily by females within the CTA-resistant phenotype. This distinction is particularly relevant given our inability to detect sex differences when data was averaged across the entire group (i.e., collapsed across phenotypes). Of note, conflicting results have been reported in prior studies with respect to the presence or absence of sex differences in ethanol-induced CTA. For example, some studies report similar ethanol- or lithium chloride-induced CTA between adult male and female rodents (Glover et al., 2016; Vetter-O’Hagen et al., 2009b). On the other hand, several studies have observed greater ethanol-induced CTA in males than females, although these differences depended on a number of variables including age, strain, and housing conditions (Morales et al., 2014; Schramm-Sapyta et al., 2014; Sherrill et al., 2011; Roma et al., 2006; Roma et al., 2007). Data from the present study suggest that differences in the prevalence of CTA-resistant and -sensitive phenotypes within a given sample may contribute to these contradictory results and that sex differences in CTA magnitude may be difficult to detect when examining population means. Instead, these differences may be better appreciated by comparing males and females within distinct phenotypes.

One possible explanation for the differences we observed in sensitivity to ethanol-induced CTA is that saccharin possesses greater hedonic value in CTA-resistant than -sensitive rats. Thus, it is plausible that in CTA-resistant rats the rewarding properties associated with saccharin consumption outweighed the aversive properties associated with acute ethanol exposure. This is unlikely, however, given that rats within each sex exhibited similar levels of saccharin intake at baseline irrespective of phenotype or ethanol dose. We did, however, observe a difference in baseline saccharin intake between males and females with females consuming significantly less saccharin than males. While these findings disagree with some previous work (Valenstein et al., 1967; Lichtensteiger, 1985), sex differences in saccharin intake have not been consistently observed (e.g., Tordoff et al., 2008; Grimm et al., 2022; Bahi et al., 2018; Huang et al., 2015). The unit of measure used in each of these studies may contribute to discrepant results in addition to the schedule of fluid access (e.g., restricted vs ad libitum). Indeed, while inclination may be to correct fluid intake measures by body weight, previous work suggests that this approach is inappropriate and has significant potential to produce misleading results (Santollo & Edwards, 2011). Nevertheless, the fact that CTA-resistant females exhibited equal, if not greater, resistance to the development of ethanol-induced CTA than males despite consuming significantly less saccharin than males at baseline lends further support to the idea that hedonic value of saccharin is unlikely to be a primary contributor to CTA resistance.

It is also possible that the difference in CTA between resistant and sensitive phenotypes is not due to a difference in sensitivity to the aversive properties of ethanol, but rather reflects differential rates of learning between phenotypes. As such, CTA-resistant rats may require additional saccharin-ethanol pairings to more robustly associate saccharin consumption with the subjective effects of acute ethanol exposure. However, the slight, but significant, reduction in saccharin intake observed in most CTA-resistant rats on conditioning day 2 suggests that CTA-resistant rats were successful in making the association between saccharin consumption and acute ethanol exposure after a single pairing but simply did not find it sufficiently aversive to avoid saccharin to the same degree as CTA-sensitive rats. Moreover, data from a separate study in our lab found that the difference in saccharin consumption between phenotypes was maintained even with additional saccharin-ethanol pairings (Ramirez et al., 2023).

While differences in the level of intoxication reached could contribute to differences in CTA between phenotypes injected with the same dose of ethanol, our data suggest that this is not a primary mediator of CTA sensitivity. This was evident using behavioral signs of intoxication, which are significantly positively correlated with BEC (Glover et al., 2019; Glover et al., 2021) and were assessed immediately after ethanol administration. As expected, we observed a dose-dependent increase in behavioral signs of intoxication in male and female rats of both phenotypes. Given that the measures of intoxication we obtained are largely reflective of signs of motor impairment and sedation, which are among the properties of ethanol considered aversive (Holdstock et al., 2000; A. C. King et al., 2002, 2011; Schuckit, 1994), we considered the possibility that rats with higher intoxication scores may also exhibit greater sensitivity to ethanol-induced CTA. However, no significant relationship was observed between these two variables in male and female rats injected with 1.5 g/kg EtOH. While a significant, albeit relatively weak, correlation was observed in rats injected with 2.0 g/kg EtOH, this was not apparent at the population level suggesting that the contribution of intoxication to CTA magnitude is not particularly robust across individuals. Moreover, the idea that greater levels of intoxication promote greater sensitivity to ethanol-induced CTA was not supported in our comparisons of the relationship between CTA magnitude and BEC – a more precise measure of intoxication with greater variability across individuals. These data are in agreement with previous work suggesting that BEC is not a reliable predictor of response to ethanol’s aversive properties (Roma et al., 2006, 2007; Sherrill et al., 2011).

While survey-based studies can capture measures of rewarding and aversive effects of acute ethanol exposure in tandem in humans, separate assays are required to assess the full range of ethanol’s properties in rodents. The present study was restricted to measurements of the response to ethanol’s aversive properties. Although an inverse relationship has consistently been observed between ethanol-induced CTA and voluntary ethanol intake, these studies have consisted largely of between-subject comparisons and ethanol drinking was not measured in the rats used in the present study. This is due, in part, to difficulty performing within-subject comparisons due to confounding effects of sequential testing of CTA and ethanol drinking. In addition, clinical work has shown that individuals at greatest risk for heavy drinking and AUD diagnosis exhibit a low response to ethanol’s aversive properties in combination with a high response to its rewarding properties (A. King et al., 2021; A. C. King et al., 2011, 2014, 2019b). Thus, it is possible that CTA-sensitive rats are equally (or more) sensitive to the rewarding properties of ethanol further complicating the ability to detect a direct relationship between CTA magnitude and drinking without additional data measuring rewarding properties in the same subjects. Related, a large body of work has characterized phenotypic differences in incentive salience toward cues predictive of rewards including ethanol. This work has shown that a subset of rats, referred to as sign-trackers, attribute incentive salience to reward-predictive cues, as evidenced by more time spent interacting with the cue. This is in contrast to goal-trackers, which respond more to the location of reinforcer delivery (Angelyn et al., 2021; Flagel et al., 2007; Flagel & Robinson, 2017; Robinson & Flagel, 2009; Valyear et al., 2017; Villaruel & Chaudhri, 2016). Importantly, the sign-tracking phenotype is associated with measures indicative of greater risk for relapse to ethanol and other drug seeking in both humans and rodent models (Cofresí et al., 2022; Colaizzi et al., 2020; Flagel et al., 2010; Robinson et al., 2014; Saunders et al., 2014). Interestingly, sign trackers are significantly more resistant to lithium chloride-induced reinforcer devaluation (i.e., CTA) than goal-trackers (Colaizzi et al., 2020; Kuhn et al., 2022). Although speculative, these data suggest the possibility that a subset of rodents exists that exhibit both a disproportionately high response to cues predictive of reward while also exhibiting resistance to devaluation with an aversive stimulus. Additional studies performing systematic and comprehensive phenotypic assessment of individual differences across multiple paradigms that interrogate both rewarding and aversive properties of ethanol and further link these differences with voluntary ethanol intake will be crucial in determining the neurobiological mechanisms underlying such differences.

Rats in the present study were classified as CTA-sensitive or -resistant based on a median split of percent change values. In doing so, all rats are assigned a phenotype even if their behavioral response is only marginally different from the median. Of note, we observed significant between-group differences despite inclusion of rats close to the median in each phenotype and replicated this effect in a second cohort of rats highlighting the robustness of our findings. Nevertheless, inclusion of rats whose behavioral response lies close to the median may be disadvantageous when examining the neurobiological mechanisms underlying these two distinct phenotypes. Thus, use of a tertile or quartile split may be a more advantageous to consider in future mechanistic studies.

In summary, the present study identifies innate differences in sensitivity to ethanol-induced CTA in outbred adult male and female Long-Evans rats. In doing so, our findings provide a much-needed animal model for studying the neural mechanisms underlying variability in subjective response to the aversive properties of ethanol. Using this model, future studies have the potential to provide crucial insight into the neurobiological factors that confer risk for heavy alcohol drinking and AUD.

Acknowledgements

We would like to thank current and past members of the Glover Lab for their technical assistance including Christen Amegashie, Alex Brown, Nidhi Chetan, Kacey Clayton-Stiglbauer, Karl Bosque-Cordero, Andres Gascon, Shikun Hou, Nikki Kinarasri, Autumn Ollice, Arleen Perez Ayala, Jacqueline Sanchez, Shree Srinivasan, and Shannon Wheeler. This work was supported by NIH grants R01 AA029130 (EJG), P50 AA022538 (EJG), R00 AA024208 (EJG), T32 AA026577 (KRP).

Support:

This work was supported by NIH grants R01 AA029130 (EJG), P50 AA022538 (EJG), R00 AA024208 (EJG), T32 AA026577 (KRP).

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