
==== Front
Drug Alcohol Depend Rep
Drug Alcohol Depend Rep
Drug and Alcohol Dependence Reports
2772-7246
Elsevier

S2772-7246(24)00061-1
10.1016/j.dadr.2024.100277
100277
Full Length Report
Effects of isolation stress and voluntary ethanol exposure during adolescence on ethanol and nicotine co-use in adulthood using male rats
Shaykin Jakob D. a
Olyha Lydia N. a
Van Doorn Catherine E. b
Hales Joshua D. a
Chandler Cassie M. a
Hopkins Deann M. b
Nixon Kimberly c
Beckmann Joshua S. a
Pauly James R. b
Bardo Michael T. mbardo@uky.edu
a⁎
a Department of Psychology, University of Kentucky, Lexington, KY 40536, USA
b Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, USA
c Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, TX 78712, USA
⁎ Correspondence to: University of Kentucky, 741 S. Limestone, Lexington, KY 40536-0509, USA. mbardo@uky.edu
21 8 2024
9 2024
21 8 2024
12 10027731 5 2024
30 7 2024
15 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background

Alcohol use in adolescence may increase susceptibility to substance use disorders (SUDs) in adulthood. This study determined if voluntary ethanol (EtOH) consumption during adolescence, combined with social isolation, alters the trajectory of EtOH and nicotine intake during adulthood, as well as activating brain neuroinflammation.

Methods

Adolescent male isolate- and group-housed rats were given 0.2 % saccharin/20 % EtOH (Sacc/EtOH) or water using intermittent 2-bottle choice; controls were given water in both bottles (n=17–20 per group). Some rats from each group (n=5–6) were euthanized one week later to measure autoradiographic [3H]PK-11195 binding, an indicator of microglial reactivity, and the remainder (n=11–14 per group) were tested in adulthood in 2-bottle choice, followed by nicotine self-administration using an incremental fixed ratio (FR) schedule with Sacc/EtOH and water concurrently available.

Results

Isolation housing increased adolescent intake of Sacc/EtOH, but the increase did not produce an observable neuroimmunological response in brain. Adolescent EtOH exposure decreased adult intake of both Sacc/EtOH and unsweetened EtOH, with isolate-housed rats showing a greater effect than group-housed rats. In the co-use model, a cross-price economic demand analysis revealed a substitutional relationship between Sacc/EtOH and nicotine, but no effect of adolescent Sacc/EtOH exposure. Compared to group-housed rats, isolate-housed rats were more sensitive to the changing price of nicotine and showed greater substitutability of Sacc/EtOH for nicotine.

Conclusion

The current results suggest that adolescent EtOH exposure per se, with or without isolation stress, does not likely explain the enhanced risk for either alcohol or nicotine use later in life.

Highlights

• Isolation stress in adolescence increases voluntary sweetened ethanol consumption.

• Voluntary ethanol intake in adolescence does not activate a neuroimmune response.

• Voluntary ethanol intake in adolescence decreases adult EtOH drinking.

• Isolation stress increases adult nicotine self-administration.

• Alcohol serves as an economic substitute for nicotine.

Keywords

Ethanol
Adolescence
Isolation
Nicotine
Economic Demand
Microglia
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pmc1 Introduction

Initiation of alcohol use often begins in adolescence and is associated with higher susceptibility of developing alcohol use disorder (AUD) in adulthood (Dawson et al., 2008, DeWit et al., 2000). As a double risk, the concomitant use of alcohol and nicotine represents the most widespread polysubstance use in the world. Over 50 % of people with severe AUD also have a tobacco use disorder, [TUD; (Weinberger et al., 2019)] and people who drink heavily are up to 3 times more likely to smoke compared to people who drink lightly (Grant et al., 2015).

Several preclinical studies have exposed rats or mice to ethanol (EtOH) in adolescence and assessed EtOH drinking in adulthood, with mixed results. With experimenter-delivered EtOH in adolescence, some studies have shown an increase in adult drinking (Maldonado-Devincci and Kirstein, 2020, Pandey et al., 2015, Sakharkar et al., 2019, Alaux-Cantin et al., 2013, Pascual et al., 2009), while others have not (Gilpin et al., 2012, Wooden et al., 2023, Broadwater et al., 2011). Similarly, with voluntary EtOH drinking in adolescence, some studies have shown an increase in adult drinking (Moaddab et al., 2017, Wooden et al., 2023, Amodeo et al., 2017, Moore et al., 2010), while others have not (Tambour et al., 2008, Vetter et al., 2007, Williams et al., 2018, Chandler et al., 2022a). Various procedural factors likely contribute to these discrepant findings.

One methodological variable that has differed across studies is the housing condition used (individual vs group caging). Isolation housing is stressful based on its ability to increase blood corticosterone (Heidbreder et al., 2000) and socially isolated rats demonstrate greater rates of self-administration across various substances, including EtOH (McCool and Chappell, 2009, Chandler et al., 2022a, Deehan et al., 2007) and nicotine (Lee et al., 2017).

Two recent studies from our laboratory examined the effect of differential housing on adolescent and adult EtOH intake using male rats. In one study, when EtOH was given via injection in adolescence, neither single- nor group-housed rats showed increased voluntary EtOH intake in adulthood, although the adolescent treatment produced a subtle neuroimmune response as measured by the microglia marker Iba1 (Wooden et al., 2023). In contrast, when EtOH was given via voluntary access in adolescence, isolate-housed rats showed greater EtOH intake during both adolescence and adulthood (Chandler et al., 2022a). However, this latter study did not examine whether voluntary EtOH intake in adolescence produced any neuroimmune response or altered nicotine co-use.

The main purpose of this study was to assess how voluntary EtOH exposure in adolescence, with or without isolation stress, affects adult EtOH consumption alone, as well as when co-used with nicotine. Male rats were used because they are more sensitive to the long-term behavioral effects of EtOH via oral gavage during adolescence compared to females (Matthews et al., 2023, Chandler et al., 2022b). In addition, we sought to extend upon previous laboratory findings from our laboratory using males (Wooden et al., 2023, Chandler et al., 2022a). A secondary purpose of this study was to assess whether adolescent EtOH exposure exacerbates EtOH-induced neurodegenerative processes in adulthood through microglial “priming” (Frank et al., 2012, Perry and Holmes, 2014, Weber et al., 2015). Evidence indicates that microglia reactions are a key cellular indicator of downstream tissue damage or cell death in the CNS (DiSabato et al., 2016, Tyler et al., 2019). Microglial reactivity is associated with the upregulation of the 18-kDa translocator protein (TSPO), a known biomarker of neuroinflammation (Guilarte, 2019, Tyler et al., 2019, Guilarte et al., 2022). In the current study, we used the radioligand [3H]-PK11195 as an indicator of microglial reactivity shown previously to be sensitive to subtle insults from EtOH (Marshall et al., 2013, Wooden et al., 2023).

2 Methods

2.1 Animals

Adolescent male Sprague-Dawley rats arrived on postnatal day (PND) 21–22 (n=72) and were randomly assigned to either group or isolate housing in a temperature-controlled colony room under a 12:12 hr light/dark cycle. Water and food were available ad libitum unless noted otherwise. All procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals (8th edition, 2011) and approved by the IACUC at the University of Kentucky.

2.2 Apparatus

Group-housing consisted of 6–7 rats in a stainless-steel cage (122 × 61 x 45.5 cm) with bedding. Isolate-housing consisted of 1 rat in a stainless-steel hanging cage (17 × 24 x 20 cm) with metal grid floors without bedding. Housing conditions were initiated upon arrival and maintained throughout the experiment, except during test sessions.

For voluntary intermittent 2-bottle choice sessions, rats were transferred to single housing in standard polypropylene cages (48 × 27 x 20 cm) equipped with wire tops and bedding. Two 250 mL bottles with standard drinking spouts (Allentown, NJ) were fixed to the wire top so that the spouts could be accessed from inside the cage.

Adult drinking and nicotine co-use occurred in two-lever operant conditioning chambers (ENV-001; MED Associates, St. Albans VT). A white cue light was located above each response lever and the back panels were modified with two circular holes (2.5 cm in diameter and 2.5 cm above floor) for access to two 100 mL Richter tubes (Model 900010; Dyets, Inc., Bethlehem, PA) with lipped feeding tube holders (Model 901100; Dytes, Inc., Bethlehem PA).

2.3 Drugs

Solutions of 20 % EtOH or 0.2 % saccharin/20 % EtOH (Sacc/EtOH) were prepared by diluting 95 % EtOH (190 proof, EtOH Pharmaco-AAPER, Shelbyville, KY) in distilled water and adding saccharin sodium salt hydrate (SIGMA-Aldrich, St. Louis, MO). Nicotine hydrogen tartrate (Sigma-Aldrich, San Diego, CA) was dissolved in 0.9 % NaCl, with dosage based on freebase weight; NaOH was added to obtain a pH of 7.4 ± 0.05.

2.4 Procedures

2.4.1 Phase 1: adolescent 2-bottle choice

Rats (PND 28–61) from each housing condition underwent three, 24-hr 2-bottle choice sessions per week (MWF) with either Sacc/EtOH or water for 15 sessions (Amodeo et al., 2017). Isolate- and group-housed rats received a bottle of Sacc/EtOH and a bottle of distilled water, or two bottles of water (n=17–20 per group). Rats were restricted to 20 g of food during the sessions and were allowed access to food and water ad libitum in their isolate or group housing condition. Bottle placement was counterbalanced and bottles were weighed before and after each session. Two empty cages with a bottle of Sacc/EtOH and water accounted for evaporation and incidental spillage from any cage rack movements.

2.4.2 [3H]PK-11195 autoradiography

A subset of isolate- and group-housed rats (n=22; n=5–6 per group) from each adolescent treatment group were euthanized by rapid decapitation one week after termination of Phase 1 drinking. Autoradiography was conducted as previously described (Pauly et al., 1989). Following decapitation, brains were removed, hemisected, and frozen in isopentane. Brains were sliced at 16 μm on a Leica CM 1850 cryostat beginning at the prefrontal cortex through the hippocampus and sections were mounted on Superfrost Plus slides. Slides were thawed, incubated in 50 mM Tris HCl (pH=7.4) buffer with 1 nM [3H]PK-11195 (PerkinElmer, Boston, MA) for 2 hr followed by washes with 50 mM Tris HCl (Marshall et al., 2013, Pauly et al., 1989). After drying overnight, slides were exposed to BioMax film (Kodak, Rochester, NY) for 8 weeks, film was developed with GBX developer (Kodak), photographed using Aiseesoft Mac Screen Recorder Version 2.0.72 and analyzed using ImageJ32 (Marshall et al., 2013).

2.4.3 Phase 2: adult 2-bottle choice

The remaining isolate- and group-housed rats underwent daily operant 2-bottle choice sessions for one hr. In Phase 2a (PND 70–76), rats were given continuous access to one bottle of 20 % EtOH and one bottle of water, and in Phase 2b (PND 77–83) they received one bottle of Sacc/EtOH and one bottle of water. Bottle placement was counterbalanced and weighed before and after each session. Rats were given 20 g food for 90 min in polypropylene cages (48 ×27 x 20 cm) following each session.

2.4.4 Phase 3: adult EtOH-nicotine co-use

Animals were food restricted (PND 84) and trained to press one lever (active lever) for food (45 mg Dustless Precision Pellets, Bio-Serv, Frenchtown, NJ) using an FR1 schedule while water and Sacc/EtOH was concurrently available as in Phase 2. Food training lasted 4–6 days, with 5 g of food given after each session.

Rats were anesthetized with a ketamine (Schein, Dublin, OH)/xylazine (Akorn, Inc., Decatur, IL)/acepromazine (Boehringer Ingelheim, St. Joeseph, MO; 75, 7.5, 0.75 mg/kg) mixture given IP and implanted with jugular catheters with a cannula secured to the skull using dental acrylic and screws. Following 5–7 recovery days, rats (PND 98–137) were trained to self-administer nicotine (0.03 mg/kg/infusion) in 1-hr sessions with Sacc/EtOH and water concurrently available as in Phase 2. Pressing the active lever delivered the nicotine infusion and a 20-sec time-out period signaled by illumination of both cue lights. Operant sessions were at FR1 for 7 days, with the response requirement increasing every three sessions from FR3 to FR135 (3, 5, 8, 12, 20, 30, 45, 60, 80, 105, 135) to calculate economic demand. Rats were provided only 10–20 g food at the end of each session.

2.5 Data analysis

Statistical analyses were conducted with Prism 9.4.1 (Graph Pad Software Inc., San Diego, CA, USA), or RStudio (R Foundation for Statistical Computing, Vienna, Austria). A correction factor described above was applied to weights of each bottle to control for evaporation and spillage.

Adolescent Sacc/EtOH consumption and preference (% intake relative to water) data were analyzed with mixed-effects models, with session as a within-subject factor and housing as a between-subject factor. Unpaired t-tests and Bonferroni multiple comparisons tests were used to probe interactions. One sample t-tests were conducted to compare EtOH and Sacc/EtOH preference to a hypothetical value of 50 % to indicate preference or indifference for EtOH. Autoradiography data were analyzed by 2 ×2 ANOVA, with each region analyzed separately.

In Phase 3, Sacc/EtOH (g/kg) and nicotine consumption (mg/kg) were determined by averaging intake on the final 2 days of each FR requirement to create a single value for each rat so consumption could be compared across FR values. To determine the slope and graphically display nicotine consumption as a function of each incremental FR requirement, nicotine consumption was plotted as a function of cost using the exponential model of demand equation (Hursh and Silberberg, 2008):Q=Q0+k(e−α(Q0∙C)−1)

where Q is consumption, Q0 is consumption at zero cost, k is a scaling constant, α is demand elasticity, and C is cost. For nicotine self-administration, C was determined by the FR requirement, Q0 was the extrapolated cost at maximum consumption, and α was the rate of decline due to cost increase.

To determine the slope and graphically display the consumption of EtOH at a fixed price as a function of the changing price of nicotine, the cross-price elasticity of demand equation (Hursh and Roma, 2016) was applied:QB=Qalone+Ie−β∙CA

where Qalone is the level of demand for the constant price of commodity B at infinite price (C) for commodity A, I is the interaction constant, β is the sensitivity of commodity B consumption to the price of commodity A, and CA is the commodity cost. Demand functions were fit to data via nonlinear mixed effects modeling (NLME), where Q0, α, I, and Qalone were free parameters, while k (3.0) and β (0.00059) were global constants. Unit price was a fixed, continuous within-subjects factor, adolescent EtOH and housing were fixed between-subjects factors, and subject was a random factor. Interactions were probed with contrasts and Bonferroni adjustments were used for multiple comparisons. To assess UP50, raw values were transformed as a percentage of maximum intake across each FR requirement.

3 Results

3.1 Phase 1: adolescent 2-bottle choice

On the first session (PND 28), isolate rats weighed more than group-housed rats (t(71) = 4.01, p <0.001). Analysis of adolescent Sacc/EtOH drinking confirmed a main effect of session (F(6.068, 201.5) = 2.605, p < 0.05; Fig. 1A), with overall consumption higher on intermediate and later sessions compared to initial sessions. The effect of housing approached significance (p < 0.1). A post-hoc unpaired two-tailed t-test collapsed across sessions confirmed that isolate-housed rats consumed significantly more Sacc/EtOH overall compared to group-housed rats (t(28) =4.76, p < 0.001; Fig. 1B), but there was no difference in water consumption between groups (Fig. 1C).Fig. 1 Adolescent Sacc/EtOH consumption and preference ratio in 2-bottle choice across sessions in group- and isolate-housed male rats. A) Adolescent Sacc/EtOH consumption (g/kg) in group- (open circles) and isolate- (filled circles) housed rats over the 15 intermittent 2-bottle choice sessions. B) Adolescent Sacc/EtOH consumption (g/kg). C) Water consumption (g/kg) collapsed across the 15 2-bottle choice sessions. D) Sacc/EtOH preference ratio displayed as % relative to total fluid consumption over the 15 intermittent 2-bottle choice sessions. E) Sacc/EtOH preference ratio displayed as % relative to total fluid consumption collapsed across the 15 2-bottle choice sessions. All data presented as mean ± SEM. # indicates p < 0.05, ## indicates p < 0.01 main effect of session relative to mixed-effects analysis. * Indicates p < 0.05, *** indicates p < 0.001 relative to t-test.

Fig. 1

Expressed as a ratio of Sacc/EtOH consumption relative to total fluid consumption (Sacc/EtOH + water), water was preferred over Sacc/EtOH in both isolate- and group-housed rats. Analysis of Sacc/EtOH preference ratios confirmed a main effect of session (F(14, 463) = 2.431, p < 0.01; Fig. 1D), with preference ratio starting low, but then increasing across sessions. Collapsed across sessions, isolate-housed rats had a significantly higher Sacc/EtOH preference ratio than group-housed rats (t(28) = 2.42, p < 0.05; Fig. 1E).

3.1.1 Autoradiography

Binding of [3H]-PK11195 measured by optical density one week after the final adolescent exposure session was negligible regardless of EtOH exposure (Fig. 2), thus showing no detectable neuroinflammation.Fig. 2 Representative pseudocolored autoradiograms containing region-specific outlines. A) Anterior section (6.63 (+1.70)) in which [3H]-PK11195 binding was analyzed in the cingulate cortex, white matter area, somatosensory cortex, striatum, nucleus accumbens shell, and nucleus accumbens core. B) Posterior section (12.05 (-5.00)) in which [3H]-PK11195 binding was analyzed in the thalamus, dorsal hippocampus, ventral hippocampus, visual cortex, auditory cortex, and entorhinal cortex. The bar represents the pseudocolored Green Fire Blue Look Up Table (LUT) from ImageJ on a scale from 1 (dark)-256 (light).

Fig. 2

3.2 Phase 2: adult 2-bottle choice

Phase 2a: Analysis of adult 20 % EtOH consumption confirmed a session x adolescent exposure interaction (F(6, 276) = 2.319, p < 0.05; Fig. 3A). A Bonferroni multiple comparisons test collapsed across housing confirmed that rats exposed to EtOH in adolescence consumed significantly less 20 % EtOH than control rats, but only on session 7 (p < 0.05; Fig. 3B).Fig. 3 Adult 20 % EtOH consumption and preference ratio in 2-bottle choice across sessions in group- and isolate-housed male rats exposed to water or EtOH during adolescence. A) Adult 20 % EtOH consumption (g/kg) in group- (open symbols) and isolate- (filled symbols) housed rats exposed to water (circles) or Sacc/EtOH (squares) as adolescents over the seven 2-bottle choice sessions. B) Adult 20 % EtOH intake collapsed across housing over the seven 2-bottle choice sessions. C) 20 % EtOH preference ratio displayed as percent relative to total fluid consumption over the seven 2-bottle choice sessions. D) 20 % EtOH preference ratio displayed as percent relative to total fluid consumption collapsed across the seven 2-bottle choice sessions. All data presented as mean ± SEM. # indicates main effect of housing, p < 0.05; & indicates session x adolescent exposure interaction relative to the mixed-effects analysis, p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, relative to Bonferroni multiple comparisons test.

Fig. 3

Expressed as an EtOH preference ratio, there was a significant effect of housing (F(1, 46) = 8.334, p < 0.01), with isolate-housed rats displaying a lower EtOH preference ratio than group-housed rats. There was also a session x adolescent exposure interaction (F(6, 276) = 2.139, p < 0.05; Fig. 3C); however, using a Bonferroni multiple comparisons test, there was no difference in preference ratios between EtOH- exposed and water-exposed rats on any session.

Collapsed across session, all groups showed an overall preference for water, except for group-housed water rats (Fig. 3D). A Bonferroni multiple comparisons test confirmed that EtOH preference ratio was decreased in isolate-housed water rats compared to group-housed water rats (p < 0.001), in isolate-housed EtOH rats compared to group-housed EtOH rats (p < 0.001) and in isolate-housed EtOH rats compared to isolate-housed water rats (p < 0.01).

Phase 2b: When shifted to Sacc/EtOH, there were no significant effects on Sacc/EtOH consumption (Fig. 4A); however, a Bonferroni multiple comparisons test collapsed across session confirmed that EtOH consumption was lower in isolate-housed EtOH rats than in isolate-housed water rats (p < 0.01; Fig. 4B).Fig. 4 Adult Sacc/EtOH consumption and preference ratio in 2-bottle choice across sessions in group- and isolate-housed male rats exposed to water or EtOH during adolescence. A) Adult Sacc/EtOH consumption (g/kg) in group- (open symbols) and isolated- (closed symbols) housed rats exposed to water (circles) or Sacc/EtOH (squares) as adolescents over the seven 2-bottle choice sessions. B) Adult Sacc/EtOH consumption collapsed across the seven 2-bottle choice sessions. C) Sacc/EtOH preference ratio displayed as percent relative to total fluid consumption over the seven 2-bottle choice sessions. D) Sacc/EtOH preference ratio displayed as percent relative to total fluid consumption collapsed across the seven 2-bottle choice sessions. All data presented as mean ± SEM. # indicates main effect of adolescent exposure relative to the mixed-effects analysis, p < 0.05. **p < 0.01, relative to Bonferroni multiple comparisons test.

Fig. 4

Expressed as a Sacc/EtOH preference ratio, analysis confirmed a significant effect of adolescent exposure (F(1, 46) = 5.250, p < 0.05), with rats given EtOH/Sacc in adolescence displaying an overall lower preference ratio in adulthood than controls. There was also a session x housing interaction (F(6, 276) = 2.767, p < 0.01; Fig. 4C); however, using a Bonferroni multiple comparisons test, there was no difference in preference ratios between isolate- and group-housed rats on any session.

Collapsed across sessions, using a one sample t-test, all groups showed a significant preference for Sacc/EtOH [Group water: (t(6) = 13.16, p < 0.001); Isolate water: (t(6) = 10.42, p < 0.001); Group EtOH: (t(6) = 13.09, p < 0.001)], except for isolate-housed EtOH rats (t(6) = 1.50, p > 0.1). A Bonferroni multiple comparisons test confirmed a significant decrease in preference ratio for group-housed EtOH rats compared to group-housed water rats (p < 0.01) and for isolate-housed EtOH rats compared to isolate-housed water rats (p < 0.01; Fig. 4D).

3.3 Phase 3: adult EtOH-nicotine co-use

Food Pre-training: On the last day of food-reinforced pretraining, analysis confirmed a main effect of housing, with isolate-housed rats earning fewer pellets than group-housed rats (F(1, 46) = 5.865, p < 0.05; results not shown).

Nicotine: Analysis confirmed a housing x FR interaction (F(11, 345) = 3.259, p < 0.001), with nicotine intake being higher in isolate-housed rats than group-housed rats on FR 1, 3, 5, 8, and 12 (Fig. 5B). There was also a housing x adolescent exposure interaction (F(1, 37) = 4.375, p < 0.05), with nicotine intake being higher in isolate-housed water rats compared to group-housed water rats (Fig. 5C).Fig. 5 EtOH-Nicotine Co-Use: A) Nicotine intake (mg/kg) across conditions over each FR. B) Mean nicotine intake at each FR, collapsed across adolescent exposure groups. C) Nicotine intake for each condition collapsed across FR. D) Sacc/EtOH consumption (g/kg) across conditions at each FR. All data presented as mean ± SEM. &,&,&, indicates housing x FR interaction relative to mixed-effects model, p < 0.001; # indicates housing x adolescent exposure interaction relative to mixed-effects model, p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, relative to Bonferroni multiple comparisons test.

Fig. 5

Sacc/EtOH: Analysis of Sacc/EtOH confirmed a FR x housing x adolescent exposure interaction (F(11, 319) = 2.694, p < 0.01; Fig. 5D). While all groups started at similarly low Sacc/EtOH consumption when nicotine was contingently available on an FR1, isolate-housed rats showed greater Sacc/EtOH consumption across increasing FR requirements following exposure to EtOH in adolescence, whereas group-housed rats showed less consumption across increasing FR requirements following exposure to EtOH in adolescence.

Economic Demand Analysis: Fig. 6 displays contingent nicotine intake and continuous access Sacc/EtOH consumption as a function of change in unit price of nicotine in isolate- and group-housed rats (Fig. 6A-6D), with best-fit parameters provided in Supplemental Materials. Own-price analysis confirmed that increases in nicotine unit price decreased nicotine intake (F(1, 356) = 69.15, p < 0.001). For Q0, consumption at a hypothetical zero cost, there was a main effect of housing (F(1, 356) = 16.11, p < 0.001), with isolate-housed rats displaying greater maximum consumption of nicotine at zero cost. For α, there was a housing x adolescent exposure interaction (F(1, 356) = 6.17, p < 0.05), with isolate-housed controls being more sensitive to the changing nicotine price than group-housed controls.Fig. 6 Demand elasticity for nicotine and Sacc/EtOH: A-D) Nicotine (mg/kg) and Sacc/EtOH intake (g/kg) for group-housed EtOH treated (filled symbols) and controls (open symbols) as a function of the changing price in nicotine. E & F) Normalized intake for Sacc/EtOH (squares) and nicotine (circles) for EtOH treatment (filled line) and controls (dotted line) as a function of the changing price in nicotine in group- (E) and isolate- (F) housed animals. Values are expressed as percent of maximal intake for individual subjects and curves were derived from nicotine own-price and Sacc/EtOH cross-price equations. All data presented as mean ± SEM.

Fig. 6

Cross-price analysis confirmed that Sacc/EtOH consumption decreased as nicotine price increased (F(1, 355) = 61.00, p < 0.001), indicative of substitution. For Qalone, there was a housing x adolescent exposure interaction (F(1, 355) = 10.21, p < 0.01), with isolate-housed EtOH rats consuming the most Sacc/EtOH at highest nicotine price. For the I parameter, there was an effect of housing (F(1, 355) = 4.28, p < 0.05), with isolate-housed rats displaying greater slopes than group-housed rats, suggesting that EtOH served as a better substitute for nicotine with isolation housing.

Finally, the point of indifference at which preference was equal for nicotine and Sacc/EtOH (UP50), although not significantly different, appear to be shifted right for EtOH-treated rats compared to controls in both group- (~1000 vs. ~1250; t(9) = 1.52, p > 0.1; Fig. 6.E) and isolate-housed (~1100 vs. ~1600; t(11) = 0.075, p > 0.1 Fig. 6F) rats.

4 Discussion

The isolation-induced increase in voluntary Sacc/EtOH consumption during adolescence observed here replicates our previous work (Chandler et al., 2022a). Similarly, Wukitsch et al. (2019) found that isolate-housed adolescent male Long-Evans rats consumed more 20 % EtOH compared to pair-housed controls using an intermittent 2-bottle choice. Despite the isolation-induced increase in voluntary EtOH intake, however, the increase was insufficient to induce neuroinflammation in the current study. Examination of [3H]-PK11195 binding one week after Sacc/EtOH drinking revealed no evidence of neuroimmune activation in either housing condition across multiple brain regions.

Previous studies examining the effect of EtOH exposure in adolescence on neuroinflammation have yielded mixed results. Using a 4-day forced high-dose (5 g/kg, oral gavage) binge model in adult male rats, one study found significant [3H]-PK11195 binding across several timepoints (Marshall et al., 2013). In contrast, using an 8-day intermittent exposure regimen at a lower dose (2 g/kg, IP) in adolescent male rats, another study found no change in either microglial activity measured by Iba1 immunoreactivity or cell death measured by fluorojade B immunoreactivity (Wooden et al., 2023); however, [3H]-PK11195 binding was not quantified. Interestingly, this latter study did observe a modest astroglial activation as measured by vimentin immunoreactivity, but it was only observed in corpus callosum, not hippocampus, and it was completely resolved by adulthood. One limitation of these previous cellular studies, as well as the current study, is the use of males only. Nonetheless, taken together with the current results, it may be that activation of a neuroimmune cell response may require a higher dose and/or more prolonged EtOH exposure than that afforded by voluntary drinking. Further, other approaches such as ELISA may reveal whether more subtle microglial activation occurred in the current study or whether other markers of neuroinflammation such as proinflammatory cytokines were present.

The isolation-induced increase in Sacc/EtOH consumption observed in adolescence was not observed in adulthood, regardless of whether EtOH was sweetened or unsweetened. One interpretation of this finding is that the repeated handling and testing procedure across development into adulthood may have mitigated the effect of isolation initially observed in adolescence. In any case, the lack of effect observed in adulthood corroborates Wukitsch et al. (2019) who reported that operant responding for EtOH did not differ between isolate- and paired-housed adult male rats exposed to EtOH during adolescence. Deehan et al. (2011) also reported no significant difference in EtOH intake or preference between isolate- and group-housed alcohol-preferring (P) and non-preferring (NP) adult rats. The consistency of those previous findings and the present results occurred despite notable procedural differences, including rodent strain, the presence of saccharin in the EtOH solution and the drinking model. However, there are some contradictory reports (Deehan et al., 2007), including one from our laboratory reporting that isolation housing transiently increased EtOH consumption in adult male rats (Chandler et al., 2022a). Importantly, that latter outcome was obtained using 24-hr access, whereas the current study was a limited 1-hr session and thus it may have been less sensitive to the effect of housing. Despite these discrepancies, one interpretation of the age-dependent effect of social isolation on drinking is that social isolation is more stressful in adolescents than adults due to a greater need for socialization, thus producing a greater compensatory increase in drinking early in life.

Another key finding from this study is that adolescent exposure to Sacc/EtOH decreased subsequent adult EtOH intake, regardless of whether it was sweetened or unsweetened. The magnitude of the decrease was greater in isolate- than in group-housed rats. If the decrease was due to a long-lasting conditioned taste aversion, the greater decrease noted in isolate-housed rats may simply reflect the greater intake of Sacc/EtOH during the adolescent period. Alternatively, the long-lasting decrease in Sacc/EtOH consumption may reflect a blunting in reward sensitivity, as adolescent exposure to EtOH reduces the number of dopamine-containing neurons in the reward-relevant ventral tegmental area (Vrettou et al., 2023). In any case, despite numerous reports showing increased adult EtOH drinking following EtOH exposure in adolescence (Maldonado-Devincci and Kirstein, 2020, Pandey et al., 2015, Siciliano and Smith, 2001, Walker and Ehlers, 2009, Alaux-Cantin et al., 2013, Pascual et al., 2009), our results add to growing evidence failing to replicate this finding (Gilpin et al., 2012, Wooden et al., 2023, Broadwater et al., 2011, Chandler et al., 2022b). Since a host of methodological differences exist across these divergent studies (e.g., dose, duration, age of exposure/testing, strain, sex, etc.), further work is required to identify the critical methodological variables that determine the influence of adolescent EtOH consumption on subsequent drinking in adulthood.

As for co-use of nicotine in adulthood, Sacc/EtOH exposure in adolescence had no effect. However, compared to group-housed rats, isolate-housed rats self-administered more nicotine and were more sensitive to the increasing price of nicotine. This latter finding should be interpreted with caution, however, as isolate-housed rats displayed greater nicotine consumption under theoretical “no-cost” conditions. In one other relevant study, Lee et al. (2017) assessed oral nicotine intake in rats using 2-bottle choice and found that isolate-housed rats consumed more nicotine solution relative to group-housed rats. The current study extends that latter finding to a route of nicotine administration (i.v.) that allows more precise control of dosing, contingent delivery and rapid absorption.

Previous work demonstrated that choice for EtOH (unsweetened) and nicotine share a negative relation with one another in female P rats (Maggio et al., 2018). This relationship, expressed using the nicotine own-price and EtOH cross-price equations, demonstrated that EtOH serves as an economic substitute for nicotine. The present study replicated this substitutive relationship, in this case using sweetened EtOH vs nicotine in male Sprague-Dawley rats. Sacc/EtOH was more substitutable for nicotine in isolate-housed rats than in group-housed rats, suggesting that isolation may modulate reinforcer sensitivity and reward valuation within our closed economy co-use model. Further, isolate-housed EtOH rats displayed the greatest Sacc/EtOH consumption when nicotine price was highest, a finding that is reciprocal in nature to the 2-bottle choice results from Phase 2 showing isolate-housed EtOH rats consuming the least 20 % EtOH and Sacc/EtOH. One possible explanation is that rats may have become sensitized to the reinforcing effects of Sacc/EtOH due to repeated exposure from Phase 2 (2-bottle choice) to Phase 3 (co-use). Moreover, a third commodity was introduced in Phase 3 (nicotine), which may have altered choice and the subsequent valuation of Sacc/EtOH. Some caution is needed, however, because the present study manipulated unit price by way of altering the response requirement (FR value) for a fixed dose of nicotine, rather than varying the unit price directly via changing the dose of nicotine.

It is notable that the substitutive economic relationship observed here between nicotine and Sacc/EtOH is contradictory to findings in the clinical literature (Bickel et al., 1995). In humans, rather than showing a substitutive relationship, changing the price of either alcohol or cigarettes decreases the consumption of both commodities, indicating a complimentary relationship (Hursh and Roma, 2016). Unlike the current work in rats, however, clinical studies have used subjective self-reports rather actual measures of drug intake and the commodity offered was a tobacco cigarette rather than nicotine. It remains to be determined what economic relation would be obtained between Sacc/EtOH and nicotine in humans undergoing a self-administration preparation like that used in rats.

5 Conclusions

Social isolation increased Sacc/EtOH consumption in adolescence, but not in adulthood. The isolation-induced increase in adolescent Sacc/EtOH was insufficient to cause a detectable neuroimmune activation in brain as measured by [3H]-PK11195 binding, but it did decrease EtOH consumption in adulthood. Adult co-use of nicotine was unchanged by adolescent Sacc/EtOH consumption, although isolation housing enhanced the substitutive relation between EtOH and nicotine. These results suggest that adolescent EtOH exposure per se, with or without isolation stress, does not likely explain the enhanced risk for either alcohol or nicotine use later in life.

Funding

This work was supported by the 10.13039/100000002 National Institutes of Health [grant numbers R01 AA025591 ; T32 DA035200 ].

CRediT authorship contribution statement

Joshua D Hales: Investigation. Cassie M Chandler: Conceptualization. Catherine E Van Doorn: Writing – original draft, Investigation. Jakob David Shaykin: Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. Lydia N Olyha: Investigation. Michael T Bardo: Writing – review & editing, Methodology, Conceptualization. Joshua Beckmann: Writing – review & editing, Formal analysis. James R Pauly: Writing – review & editing. Deann M Hopkins: Investigation. Kimberly Nixon: Writing – review & editing, Funding acquisition.

Declaration of Competing Interest

None.

Appendix A Supplementary material

Supplementary material

Appendix A Supplementary data associated with this article can be found in the online version at doi:10.1016/j.dadr.2024.100277.
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