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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Alcohol. 2020 Sep 13;89:113–122. doi: 10.1016/j.alcohol.2020.09.001

Adolescent ethanol exposure and differential rearing environment affect taste reactivity to ethanol in rats

Thomas J Wukitsch 1, Theodore J Moser 1, Emma C Brase 1, Stephen W Kiefer 1, Mary E Cain 1
PMCID: PMC7722211  NIHMSID: NIHMS1637227  PMID: 32937167

Abstract

The identification and characterization of variables that influence “liking” and enhance vulnerability to repeated alcohol use are vital to understanding and treating alcohol use disorders. In the current study, we explore the influence of rearing environment and experimenter administered adolescent ethanol on the hedonic value of ethanol, sucrose, and quinine. Male and female rats were reared for 30 days starting at postnatal day (PND) 21 in either an enriched, isolated, or standard condition and received 1.5 g/kg (i.p.) 20% (w/v) ethanol or saline every other day for 12 days starting at PND 28. Thereafter, all rats had indwelling intraoral fistulae implanted and their taste reactivity to water, ethanol (5, 10, 20, 30, 40% v/v), sucrose (0.1, 0.25, 0.5 M), and quinine (0.1, 0.5 mM) was recorded and analyzed. Results indicated that enrichment elevated hedonic responding to sucrose compared to isolation and induced a stronger negative relationship between hedonic responding and ethanol concentration compared to standard conditions. Enrichment also elevated aversive responding to ethanol and quinine compared to both isolated and standard condition rats. Adolescent ethanol injections marginally reduced aversive responding to quinine. These results replicate previous findings that environmental enrichment enhances both “liking” and aversion. In addition, the current findings suggest that, while adolescent ethanol injections may blunt aversive responses to quinine, they have no effect on aversive or hedonic responding to ethanol or sucrose. Together with existing literature our results may suggest that experience with the taste of ethanol is necessary for alterations to ethanol “liking” and aversion.

Introduction

Current theories of addiction explain that neurobiological adaptation and changes to systems involved in motivation result in addiction and substance use disorders (Berridge & Robinson, 2011, 2016; Kalivas, 2009; Robinson & Berridge, 1993, 2003; Solomon & Corbit, 1974). Incentive motivation has two components: the propensity to work for a reward or its associated stimuli (“wanting” or incentive salience) and the subjective pleasure (or displeasure) garnered from the reward itself (“liking” or hedonic value) (Berridge & Robinson, 2011, 2016; Robinson & Berridge, 1993, 2003). Much of current preclinical alcohol research focuses on the mechanisms and treatment of incentive sensitization and dependence (Cofresí et al., 2019; Nona et al., 2018). There is less emphasis on the motivational factors involved in initial and early repeated ethanol-use prior to sensitization of incentive salience, such as “liking,” which may be integral to understanding the progression and etiology of alcohol use disorder (AUD). Thus, the identification and characterization of variables that influence “liking” and enhance vulnerability to repeated alcohol use are vital to developing better treatment and preventative interventions as well as understanding incentive motivation. In the current study, we explore the influence of rearing environment and adolescent ethanol on hedonic value as measured by taste reactivity to ethanol, sucrose, and quinine.

The differential rearing paradigm is an animal model commonly used to examine the neurobiological and behavioral changes associated with differences in rearing environment (Bennett et al., 1969; Renner & Rosenzweig, 1987). This paradigm involves an isolated, an enriched, and often a standard condition into which rats are placed post-weaning. Isolated rats (IC) are not handled during the rearing period and reside in hanging metal cages. In the enriched condition (EC), rats are handled daily and have several novel objects in a large cage with many conspecifics. The standard condition (SC), in which rats are pair-housed in standard shoebox cages, is frequently used as a comparison group but does not constitute a true control for all aspects of isolation or enrichment. Post-weaning social isolation is hypothesized to enhance the incentive salience of drug rewards and associated stimuli thus increasing vulnerability to incentive sensitization, while environmental enrichment during adolescence and early-adulthood is hypothesized to do the opposite and protect against incentive sensitization (Beckmann & Bardo, 2012; Brenes & Fornaguera, 2008; Gill & Cain, 2010; Kirkpatrick et al., 2013; Stairs & Bardo, 2009). In our previous work, we showed that differentially reared rats have differences in hedonic value (Wukitsch et al., 2020). IC rats showed a pattern of taste reactions consistent with reduced “liking,” reduced aversion, and reduced hedonic sensitivity, whereas EC rats had enhanced sensitivity to changes in substance concentration and enhanced “liking” and aversion. This suggests that environment alters hedonic value without prior ethanol experience. The current study extends previous results and determines whether adolescent ethanol exposure also changes hedonic value.

Exposure to 100 mg/dL blood ethanol concentrations (BEC) repeatedly during adolescence has several lasting effects on brain areas involved in reward and motivation including neuroinflammatory damage (Pascual et al., 2007), myelin damage (Vargas et al., 2014), altered basal dopamine (Pascual et al., 2009), and changes in prefrontal cortical, amygdalar, and accumbal activity (Liu & Crews, 2015). Further, adolescent ethanol induces changes in glutamatergic signaling and epigenetic profiles that are not found in adults exposed to ethanol (Pascual et al., 2009). These repeated ethanol exposures during adolescence also result in significant behavioral alterations including deficits in motor and discrimination learning (Pascual et al., 2007). There is a somewhat mixed literature with some studies finding greater ethanol consumption during adulthood after adolescent ethanol exposure (Amodeo et al., 2017; Doremus et al., 2005; Pascual et al., 2009; Spear, 2014; but see also: Gilpin et al., 2012; Labots et al., 2018). Altogether, the evidence indicates that adolescent ethanol exposure may alter incentive motivation for ethanol; however, the question of whether adolescent ethanol exposure alters the hedonic value component of motivation is still open.

Previous research looking at the effects of ethanol exposure on taste reactivity to ethanol has been performed predominantly in adults. After voluntary ethanol drinking, increased hedonic and decreased aversive responding to ethanol is observed in adult rats among strains selected for ethanol preference (alcohol preferring [P] rats) (Bice & Kiefer, 1990), and drinking (high alcohol drinking [HAD] rats)(Kiefer et al., 1995). This effect did not occur in the selectively bred strains’ respective counterparts (alcohol non-preferring [NP] and low alcohol drinking [LAD] rats) suggesting genetic factors play a key role. However, genetic factors do not account for all the observed variability. Smaller decreases in aversive responding to ethanol from pre- to post-ethanol drinking have been found consistently among other inbred and outbred strains (Bice & Kiefer, 1990; Kiefer et al., 1994; Kiefer & Badia-Elder, 1997; Kiefer & Dopp, 1989), along with increases in hedonic responding (Bice & Kiefer, 1990; Kiefer et al., 1994; Kiefer & Badia-Elder, 1997). Because no correlation between ethanol-naive taste reactions and later ethanol consumption has been found (Bice et al., 1992; Kiefer & Dopp, 1989), the changes in taste reactivity observed after voluntary drinking experience indicate the direction of causality is likely inverted: drinking seems to be altering the hedonic value of ethanol. Whether this shift in “liking” and aversion is caused by ethanol’s pharmacological effects alone or with taste-related associative learning remains unanswered.

Because adolescent ethanol exposure has many effects on the neurobiology of reward and behavior, we hypothesized that ethanol treated rats will show higher hedonic responses and lower aversive responses to ethanol than saline treated controls. Further, enrichment will protect against increases in hedonic and decreases in aversive responses to ethanol while isolation will exacerbate the effects of ethanol experience in the opposite direction. If the pharmacological effects of ethanol alone are the cause of the hedonic shift that occurs with ethanol drinking experience, those rats with involuntary adolescent ethanol experience (i.p. ethanol injections) in the current study will differ from saline treated controls. Additionally, rats with adolescent alcohol experience in the current study will show similar taste reactivity patterns to rats with voluntary ethanol experience in previous research. Conversely, if experience with the taste of ethanol is essential to the hedonic shift, we hypothesize similar taste reactivity patterns between all rats in the current study regardless of ethanol treatment.

Methods

Animals and Rearing Conditions

One-hundred thirty-one male (n = 60) and female (n = 71) Long Evans rats (Charles River Laboratories, Kingston) arrived in the lab at postnatal day (PND) 21 and were randomly assigned to rear in one of three environmental conditions: enriched (EC; n = 42), isolated (IC; n = 44), or standard (SC; n = 45) conditions for 30 days using methods previously described (Arndt et al., 2019; Wukitsch et al., 2020). ECs were group housed (8-10 per cage) in a large metal cage (60 x 120 x 45 cm) lined with bedding. The EC cage contained 14 hard plastic objects (e.g., commercially available children’s toys, PVC pipe, plastic containers, etc.). Seven of the objects were exchanged and rearranged into a novel configuration daily. ECs were removed daily for approximately 1 minute of experimenter handling. ICs were individually housed in hanging metal cages (17 x 24 x 20 cm) with steel sides and a wire mesh front and bottom and were not handled during the rearing period except to receive injections. SC rats were briefly handled during weekly bedding changes and housed two per standard shoebox cage (20 × 43 × 20 cm) with a wire rack top and bedding. During the rearing period all rats received injections of either 1.5 g/kg (i.p.) 20% (w/v) ethanol (ETOH; n = 70) or normal saline (SAL; n = 61) every other day for 12 days starting at PND 28 (Matthews & Mittleman, 2017). The dose of 1.5 g/kg was chosen to produce blood ethanol concentrations at least as high as those attained by voluntary binge-like ethanol consumption (e.g. Simms et al., 2008), although blood ethanol concentrations were not measured in the present study due to equipment failure. The selection of the 1.5 g/kg dose was supported by our unpublished preliminary data which did not indicate differences between the environmental conditions at 1.5 g/kg (Mean ± Std. Error in mg ethanol/dL blood: EC = 138.2 ± 10.1, IC = 139.0 ± 11.7, SC = 135.3 ± 10.1) after 30 min. This metabolic similarity at the 1.5 g/kg dose also aligns with our previous work in IC and SC rats (Wukitsch et al., 2019). After the rearing period, rats remained in their respective environments for the duration of the study. All rats were housed under a standard 12-hour dark:light cycle at a temperature of 21 ±1°C and humidity between 30 and 50% along with ad libitum food and water access throughout the experiment. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (2011) and was approved by the Institutional Animal Care and Use Committee of Kansas State University.

Drugs & Chemicals

Ethanol, 200 proof, undenatured (Decon Lab, Inc.; King of Prussia, PA, USA) was prepared with sterile saline for injections (1.5 g/kg.; 20% w/v, i.p.). Ethanol, quinine HCl (Tocris Bioscience; Bristol, UK), and sucrose were prepared with sterile deionized water to specific concentrations detailed below for taste reactivity. Ketamine (60-80 mg/kg; 1 mg/ml, i.p.) and diazepam (5 mg/ml; 1 mg/ml, i.p.) were diluted to dose-specific concentrations in sterile normal saline. Isoflurane gas (1-3%, i.h.; Akorn Animal Health, Lake Forest, IL) was used during anchoring of the fistula and head mount to the skull.

Taste Reactivity

At approximately PND 65, rats were deeply anesthetized with ketamine and diazepam and were maintained with isoflurane. An intraoral fistula made of polyethylene tubing (PE-60; Instech Labs; Plymouth Meeting, PA) was implanted unilaterally, anterolateral to the first maxillary molar and threaded subcutaneously to exit on top of the skull. The tubing was connected to a head mount (313-000/SPC, PlasticsOne, Roanoke, VA) and both were secured to the skull with screws and dental acrylic. Fistulas were flushed with sterile water daily to maintain viability.

Following recovery from surgery (5-7 days), all rats began habituation sessions. At the start of each session, the rat was tethered to the leash and the swivel ensured free movement of the rat in the chamber. After 3 habituation sessions (1.5 min each) each rat received one test session (one solution) daily and orofacial movements were recorded. In the event the data from a test session was unusable (e.g., syringe pump did not work, rat became disconnected from the tether), the remaining test solution was retested prior to the final water trial. Taste reactivity to water, ethanol (5, 10, 20, 30, & 40% v/v), sucrose (0.10M, 0.25M, & 0.50M), and quinine HCl (0.1mM & 0. 5mM) were performed in an order determined by a partial Latin square so that each rat received each substance concentration once. Once the orders were determined, animals from each group were yoked to each order to counterbalance any order effects across groups. Water trials were always both the first and last trials in every order. After testing occurred, rats were returned to their home cages.

Taste reactivity testing occurred during the light cycle, in a tall trapezoidal chamber as described previously (Wukitsch et al., 2020). Briefly, a high-definition video camera (1080p @ 59.94 frames per second; Cannon Vixia HF R800) faced the chamber and recorded while solutions were administered via a syringe pump (78-0100, KDScientific, Hollison, MA) connected to a 30-mL syringe. Solutions were infused at a rate of 1 mL/min through a leash and swivel arrangement comprised of a polyethylene supply tube encased in vinyl tubing with a captive collar to secure the unit to the head mount (Plastics One; Roanoke, VA).

Rats’ orofacial responses during each taste reactivity session were scored frame-by-frame by trained raters blind to the experimental conditions of the animals using BORIS (Behavioral Observation Research Interactive Software; Friard and Gamba 2016). A single trial consisted of 1.5 minutes of infusion with a scoring time of 1 min beginning after the first visible orofacial response. The specific responses recorded were categorized using criteria previously described in the literature as either hedonic (tongue protrusions and lateral tongue protrusions) or aversive (gapes, passive drips, head shakes, forelimb flails, fluid expulsions, chin rubs, and paw pushes) (Grill & Norgren, 1978; Kiefer, 1995; Spector et al., 1988; Wukitsch et al., 2020). Table 1 shows the number of animals within each condition that had complete trials for each concentration.

Table 1.

Total Animal Number (N) by Substance and Concentration

Substance
Water
 Ethanol (% v/v)
 Quinine (mM)
 Sucrose (M)
Env. Condition Initial Final 5 10 20 30 40 0.1 0.5 0.10 0.25 0.50
EC ETOH 15 8 15 14 15 17 17 11 11 17 17 16
EC SAL 13 10 13 13 13 13 13 13 11 13 13 13
IC ETOH 14 4 14 13 15 14 14 11 9 13 12 15
IC SAL 19 12 16 14 15 16 17 18 16 18 17 17
SC ETOH 17 7 15 17 17 15 14 11 10 13 13 12
SC SAL 16 13 13 12 15 16 15 14 14 13 14 13
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Note: EC: Enriched Condition; IC: Isolated Condition; SC: Standard Condition; ETOH: Ethanol treated; SAL: Saline treated

Data Analysis

All data was analyzed using the “lme4” version 1.1-21 (Bates et al., 2015), “lmerTest” version 3.1-0 (Kuznetsova et al., 2017), and “emmeans” version 1.3.4 (Lenth, 2019) packages in R (R Core Team, 2019). To determine whether differences in taste reactions existed across Concentrations of each substance and between ethanol treatment Conditions (ETOH, SAL) and Environments (EC, IC, and SC), separate linear mixed effects models (i.e., repeated measures regressions) were performed on each class of taste reaction behaviors (hedonic or aversive) for each substance (ethanol, sucrose, quinine). Each analysis had the full factorial fixed (main) effects of Concentration, Condition, and Environment and the random effect of the intercept to create a repeated measures analysis. The random effect of the slope of Concentration (i.e., interaction between the intercept and Concentration) was included for ethanol and sucrose analyses to improve estimation of the rate of change associated with Concentration. To determine whether responses to water differed between experimental groups, separate linear mixed effects models were performed on each class of taste reaction behaviors with the full factorial fixed (main) effects of Trial (initial, final), Condition, and Environment along with the random effect of the intercept. An extended number of iterations of the model were required to reach model convergence for analyses of aversive responses to ethanol. In our previous work, sex had no effect on taste reactivity to the substances tested in the current study regardless of environmental condition (Wukitsch et al., 2020). In the current study, we did not expect to find sex effects and lacked the power to effectively analyze sex for all substances, thus, sex effects were not analyzed.

Bonferroni corrected multiple comparisons and simple slopes analyses were performed where indicated and the alpha-level was set to 0.05 for all analyses.

Interrater reliability was analyzed using separate Pearson’s correlations between each pair of raters’ totals for each class of taste reaction behaviors (hedonic or aversive) across several randomly selected videos. The average correlation for hedonic (average r = .93) and aversive (average r = .94) reactions was high between raters.

Results

Ethanol:

We determined if adolescent ethanol administered via injection could alter the taste response to ethanol in adulthood. We hypothesized that rats treated with ethanol during adolescence would show higher hedonic responses and lower aversive responses to ethanol than saline treated controls. Further, enrichment will protect against these changes that result from adolescent ethanol while isolation will exacerbate the effects of adolescent ethanol exposure.

Hedonic Reactions:

Overall, EC rats displayed a strong negative relationship between hedonic responding and ethanol concentration compared to the slightly positive relationship of SC rats. However, adolescent ethanol treatment had no effect on hedonic responding to ethanol. Means and SEMs for hedonic responses to ethanol are displayed in Figure 1A, and lines of best fit are displayed in Figure 1B. The analysis of hedonic responses to ethanol revealed a significant fixed (main) effect of Concentration, F(1,85) = 7.11, p = .009, b = −0.89, that was qualified by the interaction of Concentration and Environment. While the fixed (main) effects of Environment and Condition were not significant (ps > .05), the interaction of Concentration and Environment was significant, F(2,85) = 3.51, p = .034. Post-hoc analyses of the simple slopes showed a significant difference between EC rats’ steeply declining rate of change in hedonic responding across ethanol concentrations (b = −1.99) and SC rats’ slightly inclining rate of change (b = 0.20), t(87) = 2.65, p = .029. The interactions of Concentration and Condition, Condition and Environment, as well as the three-way interaction were not significant (ps > .05). These results suggest that adolescent ethanol treatment does not affect hedonic responding to ethanol and that rearing environment affects the rate of change in hedonic responding across ethanol concentrations.

Fig. 1.

Fig. 1.

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Overall, enrichment altered the relationship between hedonic and aversive responding and ethanol concentration. However, adolescent ethanol treatment had no effect on hedonic or aversive responding to ethanol. Mean ± S.E.M. (A) Total Hedonic and (C) Total Aversive taste reactivity responses to ethanol (5-40% v/v) between Enriched (EC), Isolated (IC), and Standard Condition (SC) rats that were repeatedly administered 1.5 g/kg (i.p) ethanol (ETOH; left side) or saline (SAL; right side) during adolescence. Lines of best fit from the linear model ± S.E.M. for each group are shown separate from mean data for ease of data illustration for both (B) Total Hedonic and (D) Total Aversive taste reactivity responses to ethanol. Hedonic responses (A & B): Adolescent ethanol treatment did not affect hedonic responses to ethanol. EC rats had a steeper rate of decline in hedonic responses to ethanol than SC rats (bp < .01). Aversive responses (C & D): Adolescent ethanol treatment did not affect aversive responses to ethanol. As ethanol concentration increased, aversive responses increased (#p <.001). EC rats had greater aversive responses to ethanol compared to both IC and SC (*p < .05) rats.

Aversive Reactions:

Adolescent ethanol treatment did not influence aversive responding for ethanol and rearing environment did not affect the positive relationship between aversive responding and ethanol concentration. However, EC rats showed higher overall aversive responses to ethanol compared to both IC and SC rats. Means and SEMs for aversive responses to ethanol are displayed in Figure 1C, and lines of best fit are displayed in Figure 1D. The analysis of aversive responses to ethanol revealed a significant fixed (main) effect of Concentration, F(1,87) = 43.44, p <.001, b = 0.61, such that, for every 1% increase in ethanol concentration, aversive responding increased by 0.61. The fixed (main) effect of Environment was significant, F(2,88) = 5.35, p = .006. Post hoc analyses revealed EC rats displayed more aversive responding to ethanol than both IC and SC rats ts(87-89) = 2.60-3.04, ps = .009-.033. The fixed (main) effect of Condition along with all 2-way, and 3-way interactions were not significant. These results indicate that adolescent ethanol treatment does not affect aversive responding to alcohol and that rearing environment affects overall aversive responding to ethanol, but not the rate of change in aversive responding across ethanol concentrations.

Sucrose:

To determine if the effects of adolescent ethanol exposure on hedonic responding are specific to ethanol, we examined hedonic and aversive reactions to a sweet reinforcer, sucrose.

Hedonic Reactions:

Adolescent ethanol treatment did not influence hedonic responding for sucrose and rearing environment did not influence the positive relationship between hedonic responding and sucrose concentration. However, EC rats showed higher overall hedonic responses to sucrose compared to IC rats. Means and SEMs for hedonic responses to sucrose are displayed in Figure 2A, and lines of best fit are displayed in Figure 2B. The analysis of hedonic responses to sucrose revealed a significant fixed (main) effect of Concentration, F(1,82) = 20.87, p <.001, b = 132.07, such that, for each 0.1 M increase in sucrose concentration, hedonic responding increased by 13.21. The fixed (main) effect of Environment was significant, F(2,84) = 5.07, p = .008. Post hoc analyses revealed EC rats displayed more hedonic responding to sucrose than IC rats, t(85) = 3.11, p = .008. The fixed (main) effect of Condition along with all 2-way, and 3-way interactions were not significant. These results suggest that adolescent ethanol treatment does not affect hedonic responding to sucrose and that rearing environment affects overall hedonic responding to sucrose, but not the rate of change in hedonic responding across sucrose concentrations.

Fig. 2.

Fig. 2.

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While enrichment resulted in higher overall hedonic responses to sucrose, adolescent ethanol treatment did not have any effect. Mean ± S.E.M. (A) Total Hedonic taste reactivity responses to sucrose (0.1-0.5 M) between Enriched (EC), Isolated (IC), and Standard Condition (SC) rats that were repeatedly administered 1.5 g/kg (i.p) ethanol (ETOH) or saline (SAL) during adolescence. Lines of best fit from the linear model ± S.E.M. each group are shown separate from mean data for ease of data illustration for (B) Total Hedonic taste reactivity responses to sucrose. Hedonic responses (A & B): Adolescent ethanol treatment did not affect hedonic responses to sucrose. As sucrose concentration increased, hedonic responses increased (#p <.001). EC rats had greater hedonic responses to sucrose than IC rats (*p < .01).

Aversive Reactions:

As expected, the number of aversive responses to sucrose was very low. Due to the low number of aversive responses for sucrose we did not conduct additional analyses. Across all conditions and concentrations, the average (± SEM) number of aversive responses to sucrose ranged from 0.42 (±1.56) to 7.89 (±2.01).

Quinine:

Given that quinine has a bitter taste similar to ethanol, we examined the effects of adolescent ethanol exposure on hedonic and aversive responses to two quinine concentrations.

Hedonic Reactions:

In general, though there was a negative relationship between hedonic responses and quinine concentration, rearing environment and adolescent ethanol treatment did not influence hedonic responding to quinine. Means and SEMs for hedonic responses to quinine are displayed in Figure 3A, and lines of best fit are displayed in Figure 3B. The analysis of hedonic reactions to quinine revealed a significant fixed (main) effect of Concentration, F(1,68) = 9.12, p = .004, b = −55.00, such that, as quinine concentration increased by 0.1 mM, hedonic responding decreased by 5.50. The fixed (main) effects of Environment and Condition along with all 2-way, and 3-way interactions were not significant. These results suggest that neither adolescent ethanol treatment nor rearing environment influenced hedonic responding to quinine.

Fig. 3.

Fig. 3.

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While enrichment resulted in higher overall aversive responses to quinine, adolescent ethanol treatment generally had a limited effect. Mean ± S.E.M. (A) Total Hedonic and (C) Total Aversive taste reactivity responses to quinine (0.1-0.5 mM) between Enriched (EC), Isolated (IC), and Standard Condition (SC) rats that were repeatedly administered 1.5 g/kg (i.p) ethanol (ETOH) or saline (SAL) during adolescence. Lines of best fit from the linear model ± S.E.M. each group are shown separate from mean data for ease of data illustration for both (B) Total Hedonic and (D) Total Aversive taste reactivity responses to quinine. Hedonic responses (A & B): Neither adolescent ethanol treatment nor environment affected hedonic responses to quinine. As quinine concentration increased, hedonic responses decreased (#p < .01). Aversive responses (C & D): ETOH rats had marginally significantly fewer aversive responses to quinine than SAL rats (>p = .054). EC rats had greater aversive responses to quinine than IC and SC (*p < .01) rats. As quinine concentration increased, aversive responses increased (#p < .01).

Aversive Reactions:

Overall, EC rats displayed more aversive responding to quinine than both IC and SC rats. Rats that received adolescent ethanol treatment displayed marginally less aversive responding compared to controls. However, neither rearing environment nor adolescent ethanol treatment influenced the positive relationship between aversive responding and quinine concentration. Means and SEMs for aversive responses to quinine are displayed in Figure 3C and lines of best fit are displayed in Figure 3D. The analysis of aversive reactions to quinine revealed a significant fixed (main) effect of Concentration, F(1,69) = 8.79, p = .004, b = 18.40, such that, for each 0.1 mM increase in quinine concentration, aversive responding increased by 1.84. The fixed (main) effect of Environment was significant, F(2,73) = 12.65, p <.001. Post hoc analyses revealed EC rats displayed more aversive responding to quinine than IC and SC rats overall, ts(72-75) = 3.44-4.90, ps = <.001-.003. The fixed (main) effect of Condition was nearing significance, F(1,73) = 3.82, p = .054, with the ETOH group displaying less aversive responding to quinine than the SAL controls overall. None of the 2-way and 3-way interactions were significant. These results indicate that rearing environment and adolescent ethanol treatment influence aversive responding to quinine but do not influence the rate of change in aversive responses across quinine concentration.

Water:

We hypothesized that both adolescent ethanol exposure and differential rearing would not alter responding for water.

Hedonic Reactions:

Unfortunately, our cell numbers decreased from water test 1 to water test 2. However, we still compared responding across the two water trials to get a general sense if behavior changed. Importantly, hedonic responding on the first water trial was similar among rearing environments. However, EC rats increased hedonic responding from the first to the second water trial which drove the differences between EC and both IC and (marginally) SC rats on the second water trial. Adolescent ethanol treatment had no effect on hedonic responding to water. Means and SEMs for hedonic responses to water are displayed in Figure 4A. The fixed (main) effect of Trial was significant, F(1,80) = 11.58, p = .001, along with the fixed (main) effect of Environment, F(2,91) = 5.04, p = .008, but these were qualified by the significant interaction of Trial and Environment, F(2,85) = 3.51, p = .034. Planned contrasts indicated that there were no significant differences in hedonic responding between environmental groups during the first water trial. However, EC rat’s hedonic responding significantly increased from the initial to the final water trial, t(84) = 4.34, p <.001, while IC and SC rats’ hedonic responding did not. This increase drove the significant differences in hedonic responding on the final water trial between EC and IC rats, t(135) = 3.48, p = .006, and the nearly significant difference between EC and SC rats, t(136) = 2.81, p = .052. The remaining 2-way and 3-way interactions were not significant. Together, these results reveal that rearing environment affects hedonic responding to water during the final but not the initial water trial and that adolescent ethanol treatment does not affect hedonic responding to water.

Fig. 4.

Fig. 4.

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Mean ± S.E.M. While adolescent ethanol treatment did not have any effect, rearing condition altered aversive, but not hedonic, responding during the first water trial. (A) Total Hedonic and (B) Total Aversive taste reactivity responses to water from the first (1) and last (2) trial of taste reactivity testing between Enriched (EC), Isolated (IC), and Standard Condition (SC) rats that were repeatedly administered 1.5 g/kg (i.p) ethanol (ETOH) or saline (SAL) during adolescence. Hedonic responses (A): Neither adolescent ethanol treatment nor environment affected hedonic responses to water on the first water trial. EC rats increased hedonic responses from the first to the last trial (^p < .001) and displayed greater hedonic responses than IC (**p < .01) and (marginally) SC (>p = .052) rats on the last trial. Aversive responses (B): Adolescent ethanol treatment did not affect aversive responses to water. EC rats had greater aversive responses to water than IC (*p < .05) rats on the first trial and decreased their aversive responses (^p < .01) to levels similar to IC and SC rats on the last trial.

Aversive Reactions:

In general, EC rats showed greater aversion to water than IC rats on the first water trial and EC rats showed decreasing aversion from the initial to the final water trial. Adolescent ethanol treatment had no effect on aversive responding to water. Means and SEMs for hedonic responses to water are displayed in Figure 4B. The fixed (main) effects of Trial, Condition, and Environment were not significant. However, the interaction of Trial and Environment was significant, F(2,75) = 5.22, p = .008. Planned contrasts indicated that EC rats displayed significantly more aversive responding than IC rats during the first water trial, t(132) = 3.01, p = .028. EC rat’s hedonic responding significantly decreased from the initial to the final water trial, t(80) = 3.43, p = .009, while IC and SC rats’ hedonic responding did not. There were no differences in aversive responding between environmental conditions during the final water trial. None of the remaining 2-way and 3-way interactions were significant. These results suggest that rearing environment affected aversive responding on the initial but not the final trial and adolescent ethanol treatment does not affect aversive responding to water.

Discussion

Our goal was to determine whether untasted, experimenter-administered, adolescent ethanol would alter taste reactivity to ethanol, sucrose, or quinine in adult rats reared in enrichment, isolation, or standard-housing environments. Rearing environment altered taste reactivity to ethanol, quinine, and sucrose, whereas, adolescent ethanol exposure only mildly affected taste reactivity to quinine alone. Enrichment elevated hedonic responding to sucrose compared to isolation and induced a stronger negative relationship between hedonic responding and ethanol concentration compared to standard conditions. Enrichment also elevated aversive responding to ethanol and quinine compared to both isolated and standard condition rats. Adolescent ethanol injections slightly reduced aversive responding to quinine. These results replicate our previous finding that environmental enrichment enhances both “liking” and aversion. In addition, the current findings suggest that while adolescent ethanol injections may blunt aversive responses to quinine, they have no effect on aversive or hedonic responding to ethanol or sucrose. Together with existing literature our results imply that experience with the taste of ethanol is critical for alterations to ethanol “liking” and aversion induced by ethanol experience.

The relationships between ethanol concentration and both hedonic and aversive responding in the current study were comparable to findings among ethanol-naive rats in the literature regardless of adolescent ethanol treatment. The directions of the relationships between substance concentrations and both hedonic and aversive responding suggest the taste reactivity methods are sensitive to changes in hedonic value. In accord with previous findings (Ferraro et al., 2002; Wukitsch et al., 2020), hedonic responding showed a positive relationship with sucrose concentration and a negative relationship with quinine concentration. Further, aversive responding was very low for sucrose, as predicted, and showed a positive relationship with quinine concentration.

The body of literature examining taste reactivity to ethanol has focused largely on the role of voluntary ethanol drinking experience in altering taste reactivity responding (Kiefer, 1995). Rats naive to the taste of ethanol tend to have relatively flat (weakly positive/negative) relationships between hedonic responding and ethanol concentration that tend to remain close to levels of hedonic responding to water (Bice et al., 1992; Bice & Kiefer, 1990; Kiefer et al., 1994, 1995; Kiefer & Badia-Elder, 1997; Wukitsch et al., 2019). The current results are similar to those of naive rats and contrast the positive relationship between hedonic responding and ethanol concentration that tends to occur after voluntary ethanol drinking that rests well above levels of hedonic responding to water (Bice & Kiefer, 1990; Kiefer et al., 1994, 1995; Kiefer & Badia-Elder, 1997). In terms of aversive responding, ethanol-naive rats tend to show a pattern of flat or weakly increasing relationships between aversive responding and ethanol concentration. In addition, the amount of aversive responding tends to remain similar to or exceed amounts of aversive responding to water (Bice et al., 1992; Bice & Kiefer, 1990; Kiefer et al., 1994; Kiefer & Badia-Elder, 1997; Wukitsch et al., 2020; but see: Kiefer et al., 1995). This ethanol-naive pattern of aversive responses also aligns with the current results. Additionally, the current pattern of aversive responses contrasts with that of rats with voluntary ethanol experience that tend to show relatively flat or negative relationships with ethanol concentration and tend not to exceed aversive responding to water (Bice & Kiefer, 1990; Kiefer et al., 1994, 1995; Kiefer & Badia-Elder, 1997).

While intraperitoneal injections are not the preferred method for intermittent ethanol administration, ethanol introduced by oral gavage could have been tasted and therefore confound any claims concerning ethanol’s pharmacological effects alone (Randich & LoLordo, 1979). Assuming the pharmacological effects of ethanol are responsible for ethanol experience-induced shifts in hedonic and aversive responding to ethanol (hedonic value shift), it is very unlikely that the ethanol injections or schedule of administration failed to result in high enough blood ethanol concentrations (BECs) to change hedonic value. In the past, i.p. injections of 2 g/kg of ethanol during adolescence reduced the voluntary consumption of sweetened ethanol during adulthood to near-zero levels (Gilpin et al., 2012). The lack of the effect of adolescent ethanol on aversive responding in the current study appears to contradict the previous finding (Gilpin et al., 2012) when taken at face value. However, in the previous study, the ethanol injections were given immediately after 30 minutes of access to the sweetener used to sweeten the ethanol during voluntary drinking in adulthood (Gilpin et al., 2012). The same methods (with sucrose instead of glucose and saccharin) have been used to induce a conditioned taste aversion (CTA) to sucrose with ethanol doses of 1.5 g/kg and higher in adolescent rats (Anderson et al., 2010). Thus, the ethanol-induced drinking reduction observed by Gilpin and colleagues (2012) is very likely due to the induction of a CTA to the sweetener rather than a shift in ethanol’s hedonic value due to the pharmacological effects of ethanol itself. This suggests that 1.5 g/kg ethanol injections are sufficient to induce changes to hedonic value only when paired with a tastant (Anderson et al., 2010). Further, when looking at voluntary consumption, continuous access conditions (1.75-3.5 g/kg/24hr) have also induced hedonic value shift in the past (Kiefer et al., 1994; Kiefer & Badia-Elder, 1997). Unfortunately, the group housing of our EC and SC conditions precluded the use of continuous or intermittent voluntary access. More research examining taste reactivity after intermittent schedules of voluntary ethanol is required to determine whether schedule of ethanol administration differentially alters hedonic value. However, given that voluntary intermittent access models result in faster escalation of intake and faster attainment of dependence than continuous access models (e.g. Kimbrough et al., 2017), intermittent access models appear more likely to exacerbate rather than attenuate hedonic value shifts when compared to continuous access. Therefore, the chosen dose and schedule of the current study should have been sufficient to induce a hedonic value shift, provided that ethanol’s pharmacological effects are the sole cause of the hedonic value shift observed in previous literature.

In the current study, adolescent ethanol treatment did not alter taste reactivity to ethanol and rats displayed patterns of taste reactivity to ethanol similar to ethanol-naive rats in previous literature. Further, in the current and previous study (Wukitsch et al., 2020) initial taste reactivity to water differed from that of the final water trial, which occurred after brief exposure to the other tastants, including ethanol. The current results also indicate that the same dose of ethanol which induces CTA (Anderson et al., 2010) does not induce a shift in hedonic value when unpaired with a tastant. When combined with the evidence concerning administration routes, schedules, and their resulting BECs, it appears the pharmacological effects of ethanol alone are not enough to shift taste reactivity to ethanol. While involuntary and voluntary ethanol consumption’s effects on taste reactivity were not directly compared in the current experiment, together, these findings further support that associations between ethanol’s taste and post-consumption effects are likely involved in changes to ethanol “liking” and aversion induced by ethanol experience. Additionally, this finding further highlights the importance of ethanol’s properties as a tastant in substance abuse research and encourages future research exploring the relationship between voluntary ethanol consumption and subsequent ethanol “liking” and aversion.

One interesting and perhaps spurious finding was that adolescent ethanol induced a marginal reduction in quinine aversion. Given that there is some similarity between quinine and ethanol as taste stimuli (Di Lorenzo et al., 1986; Lemon & Smith, 2005), one would also expect a reduction in aversive responding to ethanol either alone or in combination with a reduction in aversion to quinine. Conversely, conditioned taste aversion to ethanol does not generalize to quinine alone (Di Lorenzo et al., 1986; Kiefer & Lawrence, 1988; Kiefer & Mahadevan, 1993) highlighting differences between quinine and ethanol as stimuli that may partially explain this finding. Given the size of this effect, it would be prudent to replicate prior to further exploration or interpretation.

The current research replicates many previous findings concerning the influence of rearing environment on hedonic value (Wukitsch et al., 2020). Enrichment again caused a stronger decline in the hedonic response function across ethanol concentration compared to standard housed rats indicating higher sensitivity to changes in ethanol’s hedonic value as concentration changes. Additionally, enriched rats again had higher hedonic responding to sucrose along with higher overall aversion to ethanol and quinine compared to isolated rats indicating a shift in hedonic and aversive setpoints between isolated and enriched rats. Novel findings from the current study show enriched rats had greater aversion to ethanol and quinine than standard housed rats. This effect was trending for quinine in our previous study (Wukitsch et al., 2020). Notably, enriched rats did not have higher hedonic responding to ethanol or quinine compared to isolated rats overall as they did in our previous study (Wukitsch et al., 2020). However, the handling necessary for performing injections in the current experiment may have mitigated the isolation effect (Fone & Porkess, 2008; Holson et al., 1991; Pritchard et al., 2013; Walker et al., 2019), and/or the effect of the standard condition. The number of rats per SC cage, the size of the SC cage, and the amount of handling of the SC rats each independently alter both the neurochemical and behavioral outcomes (Renner & Rosenzweig, 1987). To truly determine the effect of handling on taste reactivity for both the IC and SC rats we would need a no-handle control group. Importantly, while the current experiment cannot directly determine the impact of handling on taste, it is clear that adolescent ethanol exposure via injection does not shift hedonic responding for a variety of tastants.

Although, isolated and standard condition rats both displayed lower levels of aversion to ethanol and quinine than enriched rats, it is worth restating that standard conditions are not a true control for isolation or enrichment and serve only as a comparison group. However, the finding that enriched rats had greater aversion to water on the first trial than isolated rats complicates interpretation. Indeed, water has gustatory properties of its own and is not a truly neutral, flavorless baseline (Rosen et al., 2010), but, because daily flushing of the fistulae were performed with the same water, it was a familiar taste stimulus compared to other tastants in the study and aversion toward it has been similar between environmental conditions in the past (Wukitsch et al., 2020). Given the low amount of aversive responding to water overall, this effect seems likely to be spurious due to a floor effect, but regardless, the direction of this initial water trial effect remains in accord with a theme of heightened aversion overall among enriched rats.

Given that CTA was induced by the same ethanol dose and administration route as was used in the current study (Anderson et al., 2010) it appears that associations between ethanol’s taste and its post-ingestive effects may be crucial to changes in hedonic value which result from ethanol experience. Early-life rearing environment impacts hedonic value in robust ways and appears to be driven most consistently by enrichment-related enhancements in aversion, isolation-related attenuation of aversion, or a combination of the two. Changes in “liking,” however, may be more sensitive to handling when isolated rats are involved, but remain consistent for the stereotypically “liked” tastant, sucrose. In light of these findings, future research should adopt a more integrative approach to investigating the neurobiology of incentive motivation for ethanol and other substances that looks at “liking,” “wanting,” and associative learning with context and interpretation that include the sensory properties of the rewards involved.

Highlights:

  • Enrichment elevated hedonic responding to sucrose compared to isolation conditions.

  • Enrichment elevated aversion to ethanol & quinine.

  • Adolescent ethanol injections marginally reduced aversive responding to quinine.

  • Adolescent ethanol injections did not affect taste reactions to sucrose or ethanol.

Acknowledgments

The authors would like to acknowledge the following undergraduate researchers whose extraordinary efforts made the current study possible: Mykenzi Allison, Robert Campbell, Chase Cunningham, Justin DePauw, Joanne Gomendoza, Natalie Kokjer, Lilly Marshall, Jared Rack, P. Mateo Small, and Riley Stearns.

Sources of Support

Research reported in this publication was supported by Kansas State University and the Cognitive and Neurobiological Approaches to Plasticity (CNAP) Center of Biomedical Research Excellence (COBRE) of the National Institutes of Health under grant number P20GM113109. The funding source had no direct involvement in the conception, data collection, interpretation, or writing of the current report.

Footnotes

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Declarations of Interest

None.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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