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. Author manuscript; available in PMC: 2024 Nov 1.
Published in final edited form as: Pharmacol Biochem Behav. 2023 Oct 5;232:173655. doi: 10.1016/j.pbb.2023.173655

Adolescent intermittent alcohol exposure produces strain-specific cross-sensitization to nicotine and other behavioral adaptations in adulthood in C57BL/6J and DBA/2J mice

Laurel R Seemiller 1, Prescilla Garcia-Trevizo 1, Carlos Novoa 1, Lisa R Goldberg 1, Samantha Murray 1, Thomas J Gould 1
PMCID: PMC10995114  NIHMSID: NIHMS1936459  PMID: 37802393

Abstract

Adolescent alcohol exposure is associated with lasting behavioral changes in humans and in mice. Prior work from our laboratory and others have demonstrated that C57BL/6J and DBA/2J mice differ in sensitivity to some effects of acute alcohol exposure during adolescence and adulthood. However, it is unknown if these strains differ in cognitive, anxiety-related, and addiction-related long-term consequences of adolescent intermittent alcohol exposure. This study examined the impact of a previously validated adolescent alcohol exposure paradigm (2–3 g/kg, i.p., every other day PND 30–44) in C57BL/6J and DBA/2J male and female mice on adult fear conditioning, anxiety-related behavior (elevated plus maze), and addiction-related phenotypes including nicotine sensitivity (hypothermia and locomotor depression) and alcohol sensitivity (loss of righting reflex; LORR). Both shared and strain-specific long-term consequences of adolescent alcohol exposure were found. Most notably, we found a strain-specific alcohol-induced increase in sensitivity to nicotine’s hypothermic effects during adulthood in the DBA/2J strain but not in the C57BL/6J strain. Conversely, both strains demonstrated a robust increased latency to LORR during adulthood after adolescent alcohol exposure. Thus, we observed strain-dependent cross-sensitization to nicotine and strain-independent tolerance to alcohol due to adolescent alcohol exposure. Several strain and sex differences independent of adolescent alcohol treatment were also observed. These include increased sensitivity to nicotine-induced hypothermia in the C57BL/6J strain relative to the DBA/2J strain, in addition to DBA/2J mice showing more anxiety-like behaviors in the elevated plus maze relative to the C57BL/6J strain. Overall, these results suggest that adolescent alcohol exposure results in altered adult sensitivity to nicotine and alcohol with some phenotypes mediated by genetic background.

1. INTRODUCTION

Adolescent alcohol use, especially binge alcohol consumption, is a national health concern. In the United States, 29% of high school students reported consuming alcohol and 14% reported binge drinking in the past month, according to a recent survey (SAMHSA, 2020). This is concerning because adolescents may be uniquely sensitive to long-term consequences of binge drinking due to ongoing brain maturation. For example, people who use alcohol during adolescence are more likely to become alcohol-dependent and use nicotine products in adulthood (McCambridge et al., 2011; Paavola et al., 2004). This could be the result of many factors, including the effect of adolescent alcohol exposure on adult drug sensitivity and mental health. Thus, it is important to understand the specific pathways through which adolescent alcohol exposure shapes adult propensity to addiction. Preclinical models of adolescent alcohol exposure can be used to address these questions.

The long-term effects of adolescent alcohol exposure on adult addiction-related behaviors have been established in animal models. For example, adolescent alcohol exposure has been shown to alter adult alcohol and nicotine sensitivity (Boutros et al., 2016; Varlinskaya et al., 2014; Waeiss et al., 2019), anxiety-like behavior and stress responses (Pandey et al., 2015; Varlinskaya et al., 2014), and cognitive functioning (Beaudet et al., 2016; Broadwater & Spear, 2014; Crews et al., 2016; Macht et al., 2020) in rodents. However, inconsistencies have been observed among these effects, which could be partially due to genetic differences across the different species and strains used in these studies. Prior work from our laboratory and others have demonstrated that adolescent and adult drug effects vary substantially across inbred mouse strains (Linsenbardt et al., 2009; Moore et al., 2010; Portugal et al., 2012; Seemiller, Logue, et al., 2022; Seemiller et al., 2023; Seemiller & Gould, 2021). More specifically, adolescent C57BL/6J and DBA/2J differ in biological and behavioral responses to acute alcohol exposure (Brodie & Appel, 2000; Camarini & Hodge, 2004; Kerns et al., 2005; Linsenbardt et al., 2009; Seemiller & Gould, 2021). A recent study found that adolescent alcohol exposure increased adult alcohol intake in C57BL/6J and not DBA/2J mice (Wolstenholme et al., 2020). These data indicate that susceptibility to other lasting behavioral effects of adolescent alcohol exposure may also vary across C57BL/6J and DBA/2J strains. Thus, we assessed the contribution of genetic background to adult phenotypes associated with adolescent alcohol exposure in C57BL/6J and DBA/2J mice.

Adolescent alcohol use has been associated with an increased risk for adult misuse of various substances, including alcohol, nicotine, cannabis, and opioids (McCambridge et al., 2011; Paavola et al., 2004; Patton et al., 2007; Thrul et al., 2021). However, effects of alcohol exposure on nicotine use (and vice versa) are particularly interesting due to overlap in their neurobiological targets and reports of cross-tolerance (Collins et al., 1988; Lopez et al., 2001; Rinker et al., 2011). In particular, nicotinic acetylcholine receptors (nAChRs) seem to critically regulate both alcohol- and nicotine-related behaviors (Miller & Kamens, 2020). Thus, the effects of adolescent alcohol on adult alcohol and nicotine sensitivity were a specific focus of these experiments.

In this study, we examined how adolescent intermittent alcohol exposure affected adult mental health and addiction-related behaviors (fear conditioning, alcohol and nicotine sensitivity, and anxiety-like behavior) in male and female C57BL/6J and DBA/2J mice. Based on prior literature, we expected that adolescent alcohol exposure would produce an addiction-susceptible phenotype in adulthood, including cognitive impairments and alterations in mental health-related endophenotyes (Beaudet et al., 2016; Boutros et al., 2016; Broadwater & Spear, 2014; Crews et al., 2016; Macht et al., 2020; Pandey et al., 2015; Varlinskaya et al., 2014, 2020; Waeiss et al., 2019; Wolstenholme et al., 2020; Younis et al., 2019). However, we predicted that this would manifest differently across strains. We examined sex differences because prior studies have shown that males and females differ in relevant phenotypes including fear conditioning, anxiety-like behavior, and thermoregulation (Sanchez-Alavez et al., 2011; Seemiller et al., 2023; Seemiller & Gould, 2021; Varlinskaya et al., 2020; Zeid et al., 2021). A primary goal of these studies is to further advance understanding of how genetic background influences susceptibility to long-lasting drug effects.

2. METHODS

2.1. Subjects

Subjects were male and female C57BL/6J and DBA/2J mice from Jackson Laboratory (Bar Harbor, ME, USA) that arrived at our facility on postnatal day (PND) 21 +/− 3. All ages listed are approximations and represent subject ages +/− 3 days. Mice were housed in groups, with ad libitum access to food (LabDiet 5053, Lab Diet, St. Louis, MO, USA) and water. All treatments and testing occurred between 7 AM and 6 PM, which was within the light period of a 12-hour light/dark cycle. Mice that lost >20% body weight or exhibited other physical signs of poor health throughout treatments were removed from the study. The Penn State Institutional Animal Care and Use Committee approved all described procedures, which were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

2.2. Timeline of treatments and testing

Experiments were run in eight balanced cohorts of approximately 46 mice over a two-year period. Subjects arrived at the facility on approximately PND 21, underwent adolescent alcohol treatments PND 30–44, and were weighed on PND 69. After fear conditioning testing (PND 70–71), mice were re-housed and split into groups of two from their previous groups of four (except in cases of groups of three, where they were moved to new cages with the same cagemates). Half of each cohort underwent battery 1 (nicotine testing) and the other half experienced battery 2 including the elevated plus maze (EPM) and alcohol testing (EPM/EtOH testing). Timelines are represented in Figure 1.

Figure 1:

Figure 1:

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Timeline of experimental procedures. Subjects acclimated to our facility, underwent two weeks of intermittent alcohol treatment, and developed into adulthood. Adult subjects underwent either foreground or background fear conditioning, and roughly equal numbers were divided into Battery 1 (nicotine) or Battery 2 (EPM/EtOH) groups.

2.3. Intermittent alcohol treatment

Beginning on PND 30 +/− 3, subjects received 0.9% saline or alcohol (2 or 3 g/kg, i.p.; 25% EtOH in 0.9% saline) once a day, every other day, for two weeks (PND 30, 32, 34, 36, 38, 40, 42, 44). This dosing regimen was chosen to be consistent with prior literature (Beaudet et al., 2016; Oliveira-da-Silva et al., 2009) and to ensure equal dosing between strains and sexes that others have shown consume different amounts of alcohol (Juárez & De Tomasi, 1999; Rhodes et al., 2005, 2007). Subjects were weighed and doses were calculated immediately prior to all injections to ensure accurate dosing across rapid adolescent development.

2.4. Fear conditioning

Because prior literature suggested that adolescent alcohol differentially affects foreground vs background contextual fear conditioning in rats (Broadwater & Spear, 2014), both variations of fear conditioning were utilized. In delay conditioning, the auditory stimulus is often the most salient (making the context a background stimulus). Foreground conditioning describes contextual fear conditioning in the absence of the auditory cue (bringing the context to the foreground).

Background fear conditioning was a two-day procedure conducted as previously described (Gould, 2003; Gould & Lommock, 2003; Gulick & Gould, 2007, 2008; Portugal et al., 2012). On the first day, subjects were placed in a novel context and observed for two minutes (baseline period). They were exposed to two conditioned stimulus (CS; white noise; 30 sec; 85 db)-unconditioned stimulus (US; mild footshock; 0.45 mA) pairings, with the two minutes in between pairings making up the immediate observation period. On the second day, subjects were returned to the training context (contextual test) for five minutes and observed for freezing behavior, or the absence of movement aside from respiration. A reduction in freezing was interpreted as impaired contextual learning. Later in the same day, subjects were placed in a novel context and observed for fear responses three minutes prior to the CS (pre-cue period) and three minutes during the CS (cued test). Freezing behavior was quantified by Noldus Ethovision (Wageningen, Netherlands). Foreground conditioning procedures were the same as background conditioning procedures, except for the absence of the CS during training and the lack of pre-cue and cued testing periods, as previously described (André et al., 2008).

2.5. Nicotine-induced hypothermia

Hypothermic response to nicotine was assessed as previously described (Seemiller et al., 2022; Zeid et al., 2021). Baseline body temperatures were measured using a TH-5 Thermalert Monitoring Thermometer and RET-3 rectal probe (Physitemp Instruments Inc., Clifton, NJ, USA) coated with Vaseline. Subjects were then treated with nicotine hydrogen tartrate salt dissolved in 0.9% saline (i.p.; 1 mg/kg free base weight – Fisher Scientific, Waltham, MA, USA) based on prior studies showing this dose is sufficient to reveal changes in nicotine-induced hypothermia due to adolescent alcohol treatment (Lopez et al., 2001). Body temperatures were measured 5, 15, 30, and 45 minutes after treatment. The ambient room temperature was 21°C +/− 1 °C.

2.6. Nicotine-induced locomotor depression

As described previously (Seemiller et al., 2022; Zeid et al., 2021), nicotine-induced locomotor depression was measured using a three-day protocol. Subjects were given injections of saline (s.c.,0.9%) and allowed to explore an open arena (45L × 45W × 40H cm; gray Plexiglas; Harvard Apparatus, Holliston, MA, USA) for 30 minutes for two acclimation days. On the third day, subjects were given an acute dose of nicotine hydrogen tartrate salt dissolved in 0.9% saline (s.c.; 0.8 mg/kg free base weight – Fisher Scientific, Waltham, MA, USA) and allowed to explore the same area for 30 minutes. This dose is similar to that used in a previous investigation of adolescent alcohol-associated changes in nicotine-induced locomotor activity (Lopez et al., 2001). Trials were video-recorded and distance traveled was measured by Noldus Ethovision (Wageningen, Netherlands).

Locomotor activity from all days was analyzed. Activity from Day 1 was used to assess locomotor differences between groups independent of adult nicotine effects. The difference in locomotor activity between Day 2 (acclimation day) and Day 3 (nicotine test) was used to assess the change in locomotor activity due to nicotine. Raw data from Days 2 and 3 are analyzed in Supplemental Materials.

2.7. Elevated plus maze

Subjects were placed in the EPM arena (55 cm tall; opposing 30L × 7W cm open arms and 30L × 7W × 13H cm closed arms; 6L × 6W cm center; gray acrylic) in dim lighting and tracked by Noldus Ethovision (Wageningen, Netherlands) for five minutes. Percent time in open arms was calculated as time spent in any open arm/total test time (300 seconds). Distance traveled was quantified in cm. Fecal boli were also quantified after each trial.

2.8. Loss of Righting Reflex (LORR)

Subjects were treated with alcohol (4 g/kg, i.p. as in Linsenbardt et al., 2009), placed in V-shaped plexiglas troughs on their backs, and monitored for LORR. LORR was defined as being unable to right twice in 60 seconds (Linsenbardt et al., 2009). The maximum time allowed for LORR was 180 minutes, and any subject that exceeded that time was recorded as having a LORR duration of 180 minutes (Chesler et al., 2012).

Immediately after regaining the righting reflex, subjects were euthanized with CO2 and blood was collected via cardiac puncture. Samples were kept on ice before being moved to room temperature for 30 minutes. Then, samples were centrifuged for 10 minutes (3000 g, 4°C). Serum was pipetted from the top, stored at −80°C, and used for BEC analysis with Analox Alcohol Analyzer (Analox Instruments, Lunenburg, MA, USA).

2.9. Statistical analysis

Principal component analysis was conducted in R (https://www.r-project.org/) and identified three subjects as outliers that were removed from subsequent analyses. Outliers were defined as observations with principal component (PC) individual scores >3 unit-variance points in any of the first 5 PCs (cumulative variance > 85%). 22 subjects (6% of 360 total subjects) exhibited signs of poor health or were euthanized during the study and their data were excluded from final analyses. Six additional subjects that underwent LORR testing were removed from LORR analyses due to never losing their righting reflex. One subject from LORR testing did not regain its righting reflex during the testing period, so its LORR duration was counted as the maximum LORR duration (180 min) and its BEC was omitted from analyses. Final sample sizes are listed in figure legends.

Statistical analyses were conducted in SPSS v26 (IBM, Armonk, NY, USA) and figures were made in GraphPad Prism (La Jolla, CA, USA). A significance threshold of 0.05 was utilized for all analyses.

Adolescent body weight and nicotine-induced hypothermia data were analyzed using 4-way ANOVA (strain, sex, treatment, timepoint) with repeated measures (timepoint). Nicotine-induced locomotor depression data were analyzed using a generalized linear mixed model with strain, sex, and treatment as fixed effects, timepoint as repeated measures, and subject ID as a random factor.

Adult body weight, fear conditioning, EPM, and LORR data were analyzed using 3-way ANOVA (strain, sex, treatment). However, to aid visualization, strains (and sometimes sexes) have been organized into separate panels. Only treatment effects are indicated in figures to simplify visualization.

Nicotine-induced locomotor depression was assessed with a general linear model with sex, strain, treatment as fixed factors and time as repeated measures. Post hoc analyses were done using Bonferroni correction for multiple comparisons.

3. RESULTS

3.1. Body weights

Throughout adolescent treatment and adult testing, body weight was monitored to aid dosing and to evaluate alcohol’s effects on development. Analysis of adolescent body weights, measured before every adolescent saline or alcohol treatment, found main effects of all factors. A main effect of strain indicated that C57BL/6J mice weighed more than DBA/2J mice (F1,345=29.770, p<0.001), a main effect of sex showed that males weighed more than females (F1,345=255.795, p<0.001), a main effect of drug demonstrated that saline-treated subjects weighed more than alcohol-treated subjects (F2,345=450.772, p<0.001), and a main effect of timepoint indicated that weights increased over time (F3,1203=1068.256, p<0.001). Additionally, strain x treatment (F2,345=3.231, p=0.041), sex x timepoint (F3,1203=27.245, p<0.001), treatment x timepoint (F7,1203=50.386, p<0.001), strain x sex x timepoint (F3,1203=21.108, p<0.001), sex x treatment x timepoint (F7,1203=3.793, p<0.001), and strain x treatment x timepoint (F7,1203=3.913, p<0.001) interactions were found. A post-hoc Tukey’s HSD test found that across strains and sexes, weights of saline-treated subjects were greater than alcohol-treated subjects (2 g/kg p=0.006; 3 g/kg p<0.001) and weights of 2 g/kg EtOH-treated subjects were more than those of 3 g/kg EtOH-treated subjects (p=0.001). Follow-up 3-way ANOVA (sex, treatment, timepoint) within each strain found main effects of sex (M>F; C57BL/6J F1,175=327.657, p<0.001; DBA/2J F1,170=64.211, p<0.001), treatment (Saline>EtOH; C57BL/6J F2,175=13.894, p<0.001; DBA/2J F2,170=11.400, p<0.001), and timepoint (C57BL/6J F3,542=539.871, p<0.001; DBA/2J F4,633=529.114, p<0.001) in addition to timepoint x sex (C57BL/6J F3,542=2.842, p=0.036; DBA/2J F4,633=45.604, p<0.001) and timepoint x treatment (C57BL/6J F6,542=16.168, p<0.001; DBA/2J F7,633=38.510, p<0.001) interactions in both strains and a timepoint x sex x treatment interaction in the DBA/2J strain (F7,633=16.168, p<0.001).

Adult weights, measured one day before fear conditioning (PND 69), were also analyzed to investigate the long-term effects of adolescent alcohol exposure on body weight. A main effect of strain suggested that DBA/2J mice weighed more than C57BL/6J mice (F1,345=11.185, p<0.001), a main effect of sex showed that males weighed more than females (F1,345=759.444, p<0.001), and a main effect of treatment indicated that saline-treated subjects weighed more than adult subjects treated with alcohol in adolescence (F2,345=3.208, p=0.042). A post-hoc Tukey’s HSD test found that weights of saline-treated subjects were greater than those treated with 3 g/kg alcohol (p=0.022). Additionally, strain x treatment (F2,345=3.318, p=0.037) and sex x treatment (F2,345=3.648, p=0.027) interactions were detected. Follow-up 2-way ANOVA (strain, sex) within treatment groups found significant main effects of strain (DBA/2J>C57BL/6J, F1,119=14.104, p<0.001) and sex (M>F, F1,119=347.275, p<0.001) among saline-treated subjects, only a main effect of sex (M>F, F1,117=254.010, p<0.001) among 2 g/kg EtOH-treated subjects, and main effects of strain (DBA/2J>C57BL/6J, F1,109=4.180, p=0.043) and sex (M>F, F1,109=178.682, p<0.001) among 3 g/kg EtOH-treated subjects. In summary, adolescent alcohol exposure reduced body weights throughout adolescence and effects persisted into adulthood (Figure 2).

Fig 2.

Fig 2.

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Body weight throughout adolescent alcohol or saline treatment (A-B) and one day prior to adult behavioral testing (C-D) in adult mice. Note that alcohol treatment only occurred in adolescence. M: male, F: female. Data are shown as mean +/− SEM. n=27–31 per strain per sex per group. Asterisks indicate a main effect of treatment (Saline>EtOH) in combined strain analyses. *p<0.05 and *p<0.001.

3.2. Fear conditioning

In adulthood (PND 70), subjects underwent fear conditioning to assess the potential long-term effects of adolescent alcohol on adult learning and memory. Prior literature suggested that effects on fear conditioning varied across foreground and background contextual fear conditioning procedures (Broadwater & Spear, 2014), so half of subjects underwent foreground and half underwent background contextual fear conditioning (see methods section for details on procedures).

3.2.1. Foreground Contextual Fear Conditioning

After foreground contextual fear conditioning training, assessment of contextual freezing revealed a main effect of strain where DBA/2J mice exhibited more freezing behavior than C57BL/6J mice (F1,165=5.018, p=0.026). Although a main effect of treatment (F2,165=2.754, p=0.067) and a strain x treatment x sex interaction (F2,165=2.870, p=0.060) approached significance, no significant effects of adolescent alcohol were observed on foreground contextual fear learning.

In the baseline period, which was the first two minutes of fear conditioning training prior to tone or footshock exposure, a main effect of strain indicated that DBA/2J mice froze more than C57BL/6J mice (F1,165=258.070, p<0.001), a main effect of sex showed that males froze more than females (F1,165=5.381, p=0.022), and a main effect of treatment showed that mice treated with alcohol during adolescence froze more than those treated with saline during adolescence (F2,165=6.216, p=0.002). A post-hoc Tukey’s HSD test found that both alcohol doses increased adult baseline freezing relative to saline controls (2 g/kg p<0.05; 3 g/kg p<0.001). A strain x sex x treatment interaction was also detected in the baseline period (F2,165=3.848, p=0.023), and follow-up 2-way ANOVA (sex, treatment) within strains found a significant sex x treatment interaction in C57BL/6J (F1,83=4.692, p=0.012) mice and not DBA/2J mice. Additionally, main effects of sex (F1,83=7.288, p=0.008; males>females) and treatment (F2,83=5.293, p=0.007; adolescent EtOH>Saline) were seen in C57BL/6J mice. Follow-up 1-way ANOVA (treatment) in C57BL/6J males and females found a main effect of treatment, with alcohol-treated subjects freezing more than saline-treated subjects, in males (F2,41=5.576, p=0.007) and not females.

In the immediate period of fear conditioning training, which occurred after tone and footshock exposure, DBA/2J mice again froze more than C57BL/6J mice (F1,165=334.049, p<0.001) and a strain x sex x treatment interaction was detected (F1,165=4.860, p=0.009). Follow-up 2-way ANOVA (sex, treatment) within strains found a significant interaction of treatment and sex in C57BL/6J mice (F2,83=5.535, p=0.006) and not DBA/2J mice. Follow-up 1-way ANOVA (treatment) within male and female C57BL/6J mice found an effect of treatment, where alcohol-treated subjects froze more than saline-treated subjects, in males (F2,41=3.558, p=0.038) and not in females. Post-hoc Tukey’s HSD tests found only a significant increase in freezing due to 2 g/kg in male C57BL/6J mice (p=0.040). Overall, adolescent alcohol influenced baseline and immediate freezing, while there were no clear effects of adolescent alcohol on adult foreground contextual fear conditioning (Figure 3).

Fig 3.

Fig 3.

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Freezing in adult mice during baseline (A-B), immediate (C-D), and contextual testing (E-F) stages of foreground fear conditioning. Note that alcohol treatment only occurred in adolescence. Data are shown as mean +/− SEM. n=12–16 per strain per sex per group. Asterisks indicate a main effect of treatment (EtOH>Saline) in the combined strain analysis of baseline freezing and a strain x treatment interaction in C57BL/6J males (EtOH>Saline) for immediate freezing. *p<0.05.

3.2.2. Background Contextual Fear Conditioning

Background fear conditioning was also conducted. While main effects for strain and sex were found, no effects of adolescent alcohol treatment were observed. Analyses and data can be found in Supplemental Materials.

3.3. Nicotine-induced hypothermia

Initial body temperature was measured immediately before nicotine injections and was analyzed to examine whether adolescent alcohol exposure could affect adult body temperatures independent of adult drug exposures. Notably, this analysis revealed a main effect of sex where females had higher body temperatures than males (F1,164=28.483, p<0.001) and a main effect of treatment where subjects treated with alcohol during adolescence had higher body temperatures in adulthood than saline-treated subjects (F2,164=4.469, p=0.013). A post-hoc analysis assessing differences across doses revealed a significant increase in body temperature only among 3 g/kg alcohol-treated subjects relative to saline-treated controls (p=0.013).

Change in body temperature due to nicotine treatment was monitored up to 45 minutes after injection. Analysis of body temperature changes revealed a main effect of strain suggesting strain-specific reduction of body temperature (F1,164=18.514, p<0.001), with C57BL/6J subjects experiencing a greater reduction in body temperature than DBA/2J subjects. A main effect of sex was also detected (F1,164=11.431, p<0.001), with females experiencing a greater reduction in body temperature than males. Change in body temperature varied by timepoint (F2,342=265.613, p<0.001). Additionally, strain x timepoint (F2,342=12.840, p<0.001), strain x sex x timepoint (F2,342=3.455, p=0.030), and strain x treatment x timepoint (F4,342=4.690, p<0.001) interactions were detected, which were followed by 3-way ANOVA (sex, treatment, timepoint) for each strain separately. In C57BL/6J subjects, a main effect of timepoint indicated that severity of hypothermia varied across timepoints (F2,164=168.818, p<0.001). In DBA/2J subjects, analysis revealed a main effect of sex where females experienced greater body temperature reductions than males (F1,82=9.817, p=0.002) in addition to a main effect of timepoint (F2,177=106.713, p<0.001). A treatment x timepoint interaction was also found in DBA/2J subjects (F4,177=4.179, p=0.002), and a follow-up 2-way ANOVA (sex, treatment) within each timepoint found that adult DBA/2J mice -treated with alcohol in adolescence had a greater reduction in body temperature than DBA/2J saline-treated subjects at the 15-minute timepoint after nicotine treatment (F2,82=3.449, p=0.036). Post-hoc analysis revealed a significant difference in body temperature among DBA/2J subjects at the 15-minute timepoint specifically between the 3 g/kg alcohol-treated subjects and saline-treated subjects (p=0.021). Additionally, DBA/2J females had larger reductions in body temperature than DBA/2J males at five (F1,82=6.968, p=0.010), fifteen (F1,82=6.593, p=0.012), thirty (F1,82=10.752, p=0.002), and forty-five (F1,82=7.866, p=0.006) minutes after nicotine treatment. Importantly, these findings suggest DBA/2J-specific cross-sensitization to nicotine after adolescent alcohol exposure (Figure 4).

Fig 4.

Fig 4.

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Body temperature before (A-B) and after (C-F) acute nicotine treatment in adulthood. Note that alcohol treatment only occurred in adolescence. Data are shown as mean +/− SEM. n=13–15 per strain per sex per group. Asterisks indicate a main effect of treatment in baseline body temperature (EtOH>Saline) and a DBA/2J-specific increased hypothermia response in EtOH-treated subjects at the 15 min timepoint after nicotine treatment (from combined sex analysis).

3.4. Nicotine-induced locomotor depression

A three-day nicotine-induced locomotor depression assay was conducted to examine adult locomotor activity in an open field after a saline injection and locomotor activity changes due to an acute nicotine treatment. Baseline locomotor activity (Day 1) and nicotine-induced hypolocomotor activity (Day 3 – Day 2) are described below with complete analyses from Days 2 and 3 found in Supplemental Materials.

Analysis of locomotor activity on Day 1 revealed a main effect of time bin (F4,733=71.356, p<0.0001) and a time bin by sex interaction (F4,733=3.107, p=0.011), with females showing higher locomotor activity than males in the first 5 minutes (F1,163 =11.018, p<0.001). Additionally, a strain by treatment interaction was found (F2,163=4.329, p=0.015). To assess strain-dependent treatment effects, follow-up analyses were conducted within each strain. In the C57BL/6J strain, there was a time bin by sex by treatment effect (F8,318=2.213, p=0.026). Bonferroni post hoc analyses revealed females in the saline-treated group having higher locomotor activity than males in the first five minutes of the assay (p=0.046), as well as in the 15-minute time bin (p=0.038), and the 25-minute time bin (p=0.009). For the C57BL/6J mice treated with 3 g/kg of EtOH in adolescence, adult males had higher locomotor activity at the 15-minute time bin (p=0.038) and at the 25-minute time bin (p=0.046) when compared to adult C57BL/6J females. In the DBA/2J strain, only a main effect of time bin (F5,385=33.728, p<0.001), with the first 5 minutes being statistically significant than all other time bins (p<0.001) was detected.

A difference score was calculated to assess nicotine-induced locomotor depression in adulthood while accounting for baseline locomotor activity (Day 3-Day 2). No effect of adolescent alcohol treatment was detected. However, a main effect of time bin (F4,680=180, p<0.001) and a strain by time bin interaction (F4,681=7.45, p<0.001) was detected. Follow-up analysis within the C57BL/6J strain revealed that locomotion varied by time bin (F4,339=103.816, p=<0.001), with all time bins being statistically different from the first five minutes (p<0.001). Additionally, locomotor activity in the 10-minute time bin was statistically significant from the 15-minute time bin (p=0.036), as well as being statistically significant from the last five minutes of the assay (p=0.013). Locomotion in the 20-minute time bin was statistically significant from locomotor activity in the 25-minute time bin (p=0.002). Follow-up analysis in the DBA/2J strain also revealed a main effect of time bin (F4,308=81.235, p<0.001). Bonferroni post hoc analyses for repeated measures revealed differences in locomotion in the first five minutes of the assay when compared to all other time bins (p<0.001). Locomotor activity in the 10-minute time bin was different from 25 (p<0.001) and 30 (p<0.001) minute time bins. In the 15-minute time bin, differences were detected from 25 (p<0.001) and 30 (p<0.001) minute time bins. Locomotion in the 20-minute time bin was statistically different than 25 (p<0.001) and 30 (p<0.001) minute time bins. Lastly, locomotor activity in the 25- and 30-minute time bins were different from all other time bins (p<0.001). In summary, adolescent alcohol did not influence adult nicotine-induced locomotor depression, but strain differences in locomotion were detected (Figure 5).

Fig 5.

Fig 5.

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Adult locomotor activity in an open field after saline treatment on the first acclimation day (A-D) and change in activity due to nicotine treatment (E-H). Nicotine locomotor depression is represented by the difference in distance traveled between the second day of testing (after saline) and the third day of testing (after nicotine; see methods section for details). Data are shown as mean +/− SEM. n=13–16 per strain per sex per group. Sex by treatment interactions were detected for Day 1 locomotor activity.

3.5. Elevated plus maze

Anxiety-related behaviors were assessed in adulthood using an elevated plus maze. No effects of adolescent alcohol treatment were found, but differences in strain and sex were seen; analyses and data are available in Supplemental Materials.

3.6. Loss of Righting Reflex (LORR)

Sensitivity to alcohol’s hypnotic effects in adulthood was evaluated by measuring latency to LORR, LORR duration, and BEC at the time of LORR recovery. First, analysis of LORR latency identified a main effect of treatment (F2,160=9.460, p<0.001). Post-hoc Tukey’s HSD analysis revealed that adult mice treated with 3 g/kg alcohol during adolescence lost their righting reflex later than mice treated with saline during adolescence (p<0.001). No effects of strain or sex were detected.

Analysis of LORR duration identified a main effect of strain where DBA/2J mice had a longer LORR duration than C57BL/6J mice (F1,160=3.908, p<0.050) and a main effect of sex where males had a longer LORR duration than females (F1,160=12.256, p<0.001). A sex x strain interaction was also detected (F1,160=12.997, p<0.001), and follow-up 2-way ANOVA (sex, treatment) within each strain found a main effect of sex only in DBA/2J mice (F1,78=24.846, p<0.001), where males had a longer LORR duration than females. Notably, this analysis also detected a main effect of treatment in DBA/2J mice (F2,78=3.179, p=0.047), where DBA/2J subjects treated with alcohol during adolescence had shorter LORR duration than DBA/2J subjects treated with saline during adolescence.

Analysis of BEC at LORR recovery also revealed a strain x sex interaction (F1,159=5.221, p=0.024). To follow up on this interaction, a 2-way ANOVA (sex x treatment) within strains was performed but failed to identify any significant effects of sex or treatment. Thus, adolescent alcohol exposure increased latency to LORR but not duration of LORR or BEC at recovery of LORR (Figure 6).

Fig 6.

Fig 6.

Open in a new tab

Latency to LORR (A-B), LORR duration (C-D), and BEC at regain of LORR (E-F) after acute alcohol treatment. Note the Saline, 2 g/kg EtOH, and 3 g/kg EtOH designations refer to adolescent treatment. All mice received 4 g/kg EtOH in adulthood for LORR tests. Data are shown as mean +/− SEM. n=12–16 per strain per sex per group. Asterisks indicate a significant main effect of treatment on latency to LORR in combined strain analyses (EtOH>Saline). **p<0.001.

4. DISCUSSION

The aim of this study was to examine if the long-term effects of adolescent alcohol exposure on behavior and drug sensitivity were modulated by genetic background. Learning and memory, anxiety-related behavior, and drug sensitivity were tested in adult C57BL/6J and DBA/2J mice after adolescent alcohol or saline exposure. Adolescent alcohol exposure led to DBA/2J strain-specific increased adult sensitivity to hypothermia after an acute nicotine challenge. Adolescent alcohol exposure also increased adult latency to alcohol-induced LORR across both strains. These findings suggest selective tolerance to alcohol and cross-sensitization to nicotine due to adolescent alcohol exposure and genetic background. Adolescent alcohol exposure selectively altered freezing in some stages of fear conditioning but did not result in detected differences in anxiety-related behavior in the elevated plus maze and nicotine-induced locomotor depression. Phenotypes that were also seen in adulthood after adolescent alcohol exposure include increased freezing behavior during fear conditioning training and an increased baseline body temperature. Adolescent intermittent alcohol exposure also significantly disrupted adolescent development, as evidenced by persistent reductions in body weight among alcohol-treated subjects. Collectively, these findings indicate that genetics and sex contribute importantly to addiction-related, anxiety-related, and cognitive phenotypes. Further, adolescent alcohol exposure can have unique effects on adult drug sensitivity depending on genetic background.

First, body weight was monitored throughout adolescent development and prior to adult behavioral testing to evaluate gross developmental effects of alcohol exposure. Strain and sex produced predictable differences in body weight, where adolescent DBA/2J mice weighed less than adolescent C57BL/6J mice and males weighed more than females, as has been previously reported (Moore et al., 2011). Additionally, alcohol-treated subjects demonstrated significant reductions in body weight throughout adolescence and in adulthood. Low body weights in adulthood due to adolescent alcohol treatment have been observed previously in rodent studies (Forbes et al., 2013; Huang et al., 2012; Sherrill et al., 2011). Because alcohol is a teratogen and doses were relatively high (modeling binge-like alcohol use), this was also expected in our study. The potential association between body weight and brain development in preclinical models of adolescent alcohol exposure is unclear. One study examining low body weights associated with adolescent alcohol exposure and brain development could not identify an association between body weight and the mass of cerebral cortex, cerebellum, or corpus callosum in mice (Huang et al., 2012). Thus, the observed reduction in body weights of our adolescent alcohol-treated mice does not necessarily indicate gross neuroanatomical changes.

Adult foreground and background fear conditioning were assessed after adolescent alcohol exposure. Prior work from our laboratory (Seemiller et al., 2023; Seemiller & Gould, 2021) and our current findings have consistently shown that DBA/2J mice freeze dramatically more than C57BL/6J mice during fear conditioning training (in the baseline and immediate stages) but similarly during contextual and cued testing. This is notable and demonstrates that strain modulates the expression of fear behavior in learning-dependent and -independent contexts. We have also found that adolescent and adult C57BL/6J and DBA/2J differ in acute alcohol-associated fear conditioning impairments, and other research has shown that adolescent alcohol exposure differentially alters adult foreground and background fear conditioning (Broadwater & Spear, 2014). Specifically, they observed that contextual freezing was decreased after foreground contextual fear conditioning but increased after background contextual fear conditioning in adult rats after adolescent alcohol exposure. Thus, we hypothesized that we would observe strain- and task-dependent cognitive deficits after adolescent alcohol. However, this was not the case. No effects of adolescent alcohol exposure on contextual or cued learning were observed, regardless of foreground or background fear conditioning training conditions. Generally, studies of adult learning and memory after adolescent alcohol exposure have reported inconsistent effects, likely related to use of different rodent models (rats vs mice), heterogeneity in alcohol exposure paradigms (timing and intermittency), time in between treatment and testing, and testing conditions (Seemiller & Gould, 2020; Spear, 2020). It is possible that our choice of hippocampus-dependent learning (contextual fear conditioning) could not capture the nuanced cognitive changes caused by our adolescent alcohol exposure paradigm, as learning tasks that are more dependent upon the prefrontal cortex seem to be more consistently impaired across studies (Seemiller & Gould, 2020). Alternatively, there may be different doses of alcohol and exposure paradigms that could have produced different behavioral profiles. Further, we observed some increases in baseline and immediate freezing due to adolescent alcohol exposure across both strains, suggesting that adolescent alcohol could be altering adult fear behaviors in a manner independent of fear learning. These findings suggest that adolescent alcohol exposure may not directly affect adult fear learning, but adolescent alcohol exposure, genetic background, and sex may separately influence fear conditioning-related phenotypes.

Adolescent alcohol consumption has been associated with an increased risk for adult nicotine use (Paavola et al., 2004). One possible explanation for this association is that alcohol use changes sensitivity to nicotine, making it more reinforcing. Other preclinical literature supports this idea and has shown that developmental alcohol exposure increased dopaminergic responses to nicotine (Waeiss et al., 2019) and decreased sensitivity to rewarding effects of nicotine (Boutros et al., 2016) in adulthood. We tested whether adolescent alcohol exposure could alter adult nicotine-induced hypothermia. Prior research conducted in C57BL/6J mice has shown that adolescent alcohol exposure can reduce adult sensitivity to nicotine-induced hypothermia (Lopez et al., 2001) and, in male Sprague Dawley rats, adolescent nicotine exposure can reduce adult sensitivity to alcohol-induced hypothermia (Rinker et al., 2011). In our study, we found that adolescent alcohol exposure increased adult baseline body temperature, consistent with prior work (Lopez et al., 2001). Females also had higher body temperatures than males, which has been observed previously (Sanchez-Alavez et al., 2011) and may be related to sex differences in thermoregulation. Further, we found that nicotine-induced hypothermia effects were dependent on strain background. While not significant, our data suggest a trend in female C57BL/6J mice that recapitulates what was found in prior work, with adolescent alcohol reducing adult nicotine sensitivity in this task (Lopez et al., 2001). This inconsistency may be due to differences in alcohol treatment paradigms and delays between treatment and testing across studies. However, in our study, DBA/2J mice exhibited a significant increase in sensitivity to nicotine due to adolescent alcohol. This suggests a cross-sensitization to nicotine after adolescent alcohol exposure that is specific to the DBA/2J strain. This could indicate a broader sensitivity to aversive effects of nicotine because hypothermic effects of drugs like nicotine and alcohol have been associated, imperfectly, with their aversive properties. For example, reducing alcohol-induced hypothermia by increasing ambient temperature was sufficient to reduce conditioned aversion to alcohol (Cunningham et al., 1988). Taken with our findings, this could indicate that some genetic backgrounds (like DBA/2J) may be more prone to avoid nicotine after adolescent alcohol exposure because they are more susceptible to nicotine-induced hypothermia. More broadly, these findings reinforce the importance of genetic background in determining the long-term consequences of adolescent drug exposure. While others have demonstrated differences in nicotine sensitivity after alcohol treatment or alcohol sensitivity after nicotine treatment across mouse strains in adult subjects (de Fiebre & Collins, 1993; Slater et al., 2016), the current data establish the importance of examining genetics and the effects of adolescent alcohol exposure on adult behavior. This relationship between alcohol and nicotine may also be important when considering treatment outcomes for addiction (Rahman et al., 2015).

The effects of adolescent alcohol exposure on adult nicotine sensitivity were specific to hypothermic effects of nicotine, as no impact of adolescent alcohol was observed on locomotor depression after adult nicotine treatment. Few studies have examined adolescent alcohol’s effects on adult nicotine locomotor depression, but one study found that adolescent alcohol exposure reduces sensitivity to nicotine-induced locomotor depression (Lopez et al., 2001). In our data set, a strain-specific effect of adolescent alcohol was observed on adult locomotor activity during the first acclimation session (post-saline). This could suggest that adolescent alcohol exposure alters baseline locomotor activity in a manner that interferes with the expression of nicotine-induced locomotor depression. It is perhaps more likely that adolescent alcohol exposure produces a selective cross-tolerance to nicotine that does not affect all nicotine sensitivity measures.

Anxiety-related behaviors were assessed using an EPM. Again, no effect of adolescent alcohol exposure was seen on adult EPM behaviors. Previous work on anxiety-related phenotypes after adolescent alcohol exposure have shown varied results, ranging from increased (Varlinskaya et al., 2020) to decreased (Healey et al., 2022) anxiety-related behavior during adulthood after adolescent alcohol exposure, while other results align with our finding of no change (Torcaso et al., 2017; Younis et al., 2019). Others have pointed out these inconsistencies in anxiety phenotypes after alcohol exposure in preclinical models and have suggested they may result from inconsistent alcohol exposure paradigms and behavior testing methodologies across the described studies (Bloch et al., 2022). Cumulatively, these data suggest that the effects of adolescent alcohol on adult anxiety-related behaviors are nuanced and may be sensitive to experimental variation. In contrast to the null effects of adolescent alcohol, strain effects on EPM behaviors were very consistent with prior reports (Moore et al., 2011). DBA/2J mice consistently spent less time in open arms and produced more fecal boli, indicating a greater anxiety-like phenotype in DBA/2J mice. Thus, in this experiment, genetic background was more important than adolescent drug use in determining anxiety-like phenotypes.

Finally, to follow up on human findings describing an increased risk for adult alcohol use after adolescent alcohol exposure (McCambridge et al., 2011), we tested the influence of adolescent alcohol exposure on adult alcohol sensitivity via assessment of LORR after an acute alcohol treatment. Adolescent alcohol exposure increased the latency to LORR across both strains. This suggests some tolerance to alcohol in adulthood after adolescent alcohol exposure in a manner that is independent of strain. Decreased sensitivity to alcohol in adulthood could support elevated problematic drinking behaviors, providing a possible mechanism for human data describing a relationship between adolescent and adult use (McCambridge et al., 2011). Others have reported a similar adult tolerance to alcohol as measured by time to LORR onset (Younis et al., 2019), although this prior report found it was accompanied by longer LORR duration. It is possible that the longer latency to LORR is related to increases in baseline body temperature observed prior to the nicotine hypothermia assay in our data set, since alcohol-induced changes in body temperature may contribute to LORR. Specifically, others have reported that ambient and body temperatures can change sensitivity to alcohol-induced LORR in a strain-dependent manner (Finn et al., 1990, 1994). In our study, adolescent alcohol-treated subjects had higher baseline body temperatures and a longer latency to alcohol-induced LORR across both strains. Overall, results from this test suggest that some long-term adaptations to adolescent alcohol exposure, such as tolerance to alcohol assessed by LORR, may be consistent across strain backgrounds.

In conclusion, we conducted a series of assays (fear conditioning, nicotine hypothermia, nicotine locomotor depression, EPM, LORR, body weight) in adult C57BL/6J and DBA/2J mice after adolescent intermittent alcohol exposure. In many cases, pronounced strain and sex effects were observed independent of prior alcohol treatment, demonstrating the profound importance of genetic background in determining addiction-related, anxiety-related, and cognitive phenotypes. In addition, we found evidence of a strain-specific cross-sensitization to nicotine-induced hypothermia in DBA/2J mice and shared tolerance to alcohol-induced LORR in C57BL/6J and DBA/2J mice. This demonstrates that for some phenotypes, strain background and adolescent alcohol experience may interact to produce unique drug sensitivities in adulthood. However, not all phenotypes were altered by alcohol. Cumulatively, adolescent alcohol exposure and genetic background produced complex behavioral effects in adulthood. Our findings demonstrate that the impact of adolescent alcohol exposure on adult drug sensitivity can be dependent upon genetic background. This work calls for careful consideration of genetic background in future studies and highlights adolescence as a vulnerable period for exposure to substances of abuse.

Supplementary Material

1

Highlights.

  • Adolescent alcohol exposure is associated with lasting behavioral changes

  • Adolescent alcohol exposure increased adult sensitivity to nicotine

  • Adolescent alcohol exposure altered adult tolerance to alcohol

  • Adolescent alcohol exposure effects interacted with genetic background

ACKNOWLEDGEMENTS

We thank Dr. Helen Kamens for sharing equipment necessary for this project, and we thank Dr. Nicole Crowley for her early conceptual input. Additionally, we thank Dr. Sean Mooney-Leber for his help in troubleshooting methodology in the early stages of the project.

FUNDING AND DISCLOSURE

This study was supported by the National Institutes of Health [T32GM108563 (L.R.S.) and T32DA017629 (P.G.T.)], Fulbright Minciencias Scholarship (C.N.), the Jean Phillips Shibley Endowment (T.J.G.); and Penn State University (T.J.G, S.M.). The authors declare that they have no competing interests.

Footnotes

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