Prenatal ethanol exposure potentiates isolation-induced ethanol consumption in young adult rats

Alcohol 75 (2019) 39e46

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Prenatal ethanol exposure potentiates isolation-induced ethanol consumption in young adult rats
ndez a, 1, Jorge Carrizo b, 1, Wladimir Plaza c, Paola Haeger c, *, Macarena Soledad Ferna Ricardo Marcos Pautassi a, **
n M

rdoba, C.P 5000, Argentina Instituto de Investigacio edica M. y M. Ferreyra (INIMEC e CONICET-UNC), Co
rdoba, Co
rdoba, C.P. 5000, Argentina Facultad de Psicología, Universidad Nacional de Co c
lica del Norte, Coquimbo, Chile Departamento de Ciencias Biom
edicas, Facultad de Medicina, Universidad Cato a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 March 2018 Received in revised form 4 May 2018 Accepted 8 May 2018

Prenatal and/or early postnatal ethanol exposure (PEE) is associated with significant behavioral and physiological deficits in offspring, including alterations in stress response systems and a greater likelihood of alcohol use disorders. Stress-induced ethanol drinking after PEE, however, has been largely unexplored. The present study analyzed ethanol intake in male Sprague-Dawley rats after protracted prenatal and early postnatal ethanol exposure and tested whether social isolation during the sensitive period of adolescence modulates the effects of PEE on ethanol drinking. The dams were given 10% ethanol (or its vehicle) as the sole drinking fluid from gestational day 0 (GD0) to postnatal day 7 (PD7). On PD21, male offspring were housed individually (isolated housing group) or in pairs in standard cages (standard housing group). From PD56 to PD84, these male rats were tested for ethanol intake in 24-h, intermittent two-bottle choice sessions that were conducted across 4 weeks. Maternal ethanol consumption during gestation and during the first week of life of the offspring averaged 6.10e8.20 g/kg/22 h. Isolation housing during adolescence increased free-choice ethanol drinking in young adulthood. The main novel finding was that this facilitative effect of isolation on absolute and percent ethanol intake was significantly greater in PEE rats than in control counterparts not exposed to the prenatal and early postnatal ethanol exposure (effect sizes [h2p]: 0.24e0.32). The present results suggest that PEE renders the individual sensitive to the facilitative effect of stress exposure on ethanol intake. © 2018 Elsevier Inc. All rights reserved.

Keywords: Intake Fetal programming Adolescence Stress

Introduction Despite preventive efforts, alcohol (ethanol or EtOH) consumption during pregnancy is still highly prevalent worldwide. Studies that were conducted in South America, for example, reported a prevalence of alcohol use (any dose) during pregnancy between 50% (Hutson, Rao, Fulga, Aleksa, & Koren, 2011) and 75%
pez, Filippetti, & Cremonte, 2015). The latter study (Lo
pez et al., (Lo 2015) indicated that 15.1% of the sample (614 women, 13e44 years


dicas, Facultad de * Corresponding author. Departamento de Ciencias Biome
lica del Norte, Larrondo, 1281 Coquimbo, Chile. Medicina, Universidad Cato
n Me
dica M. y M. Ferreyra ** Corresponding author. Instituto de Investigacio
rdoba, C.P. 5016, Argentina. Fax: þ54 351 (INIMEC e CONICET), Friuli 2434, Co 4695163. E-mail addresses: [email protected] (P. Haeger), [email protected] (R.M. Pautassi). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.alcohol.2018.05.006 0741-8329/© 2018 Elsevier Inc. All rights reserved.

old) had at least one episode of binge drinking (i.e., >5 drinks in a 2h period) during pregnancy. Preclinical (Spear & Molina, 2005) and clinical (Bookstein, Streissguth, Connor, & Sampson, 2006; Donald et al., 2015) studies have shown that prenatal ethanol exposure (PEE) is associated with significant behavioral and physiological deficits in offspring, including a greater likelihood of alcohol use disorder (AUD) later in life. Prenatal ethanol exposure but not prenatal nicotine exposure predicted alcohol drinking problems at age 21 (Baer, Sampson, Barr, Connor, & Streissguth, 2003), whereas a study with adoptees found significantly greater symptoms of AUD in 21 subjects whose mothers drank during pregnancy than in 21 peers without this developmental insult (Yates, Cadoret, Troughton, Stewart, & Giunta, 1998). Our studies that utilized rat models found that even brief ethanol exposure during late gestation (GD17 to GD20, 2.0 g/kg/ day, intragastric [i.g.]) enhanced ethanol intake throughout infancy

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ndez et al. / Alcohol 75 (2019) 39e46 M.S. Ferna

(Pueta, Rovasio, Abate, Spear, & Molina, 2011) and adolescence (Fabio et al., 2013), promoted ethanol-induced conditioned preference (Pautassi, Nizhnikov, Spear, & Molina, 2012), and heightened ethanol-induced dopaminergic activity in the ventral tegmental area (Fabio, Vivas, & Pautassi, 2015). Our group and others have also employed animal models using protracted and intense ethanol exposure. For example, we found greater intake of 10% ethanol as the sole drinking fluid in late adolescent offspring of dams that were exposed to ethanol throughout pregnancy and up to postnatal day (PD) 7 (Contreras et al., 2017). The infant rat during its first postnatal week resembles the third trimester of pregnancy in humans (Dobbing & Sands, 1979). Therefore, this time frame of ethanol exposure models human fetal exposure throughout pregnancy. The study by Contreras et al. (2017) had the caveat of testing only one ethanol concentration in a single 24-h test. The mechanisms that underlie the facilitative effect of PEE on subsequent ethanol intake are still mostly unknown. Prenatal ethanol exposure appears to facilitate subsequent ethanol drinking by altering the function of N-methyl-D-aspartate receptors (Honse, Randall, & Leslie, 2003; Samudio-Ruiz, Allan, Valenzuela, PerroneBizzozero, & Caldwell, 2009) or the mesocorticolimbic dopamine system (Fabio et al., 2015). Some have suggested that the contiguity between the odor/taste of ethanol that is experienced in utero and the rewarding pharmacological effects of ethanol result in the acquisition of conditioned preference for the drug (Spear & Molina, 2005). Under this framework, subsequent re-exposure to the odor/ taste of ethanol may trigger ethanol seeking or intake. Another possibility, as discussed by Chotro, Arias, and Laviola (2007), is that PEE enhances subsequent responses to stress and stress-induced drinking. Recent evidence suggests that PEE can alter the k opioid receptor system (Nizhnikov et al., 2014), which mediates the effects of stress and reduces the acute reinforcing effects of ethanol. Stress has long been recognized as a vulnerability factor for heightened ethanol drinking, although its use in animal models has yielded highly variable outcomes (Becker, Lopez, & Doremus-Fitzwater, 2011). Ethanol exposure is also a stressor itself. The drug activates the hypothalamic-pituitary-adrenal (HPA) axis, resulting in the release of corticosterone (CORT) (Cannizzaro, La Barbera, Plescia, Cacace, & Tringali, 2010). Chronic ethanol exposure, which is akin to ethanol exposure that is employed in animal models of PEE, alters ethanol- or stress-induced activation of the HPA axis. For example, rats that were subjected to binge-like intermittent ethanol exposure exhibited greater signs of depression (akin to those induced by chronic stress), than controls after exposure to social defeat-induced stress (Boutros et al., 2017). Several preclinical studies indicate that PEE can alter stress response systems. Nelson et al. (1986) found an increase in the footshock-induced release of CORT in PEE rats compared with control rats. Other studies reported that PEE heightened basal HPA tone and altered recovery of the HPA axis after stress exposure (Haley, Handmaker, & Lowe, 2006; Weinberg, Sliwowska, Lan, & Hellemans, 2008). In another study, male and female offspring of dams that were exposed to ethanol through an ethanol liquid diet (resulting in intake of 14e16 g/kg/day) were subjected to chronic variable stress (i.e., a paradigm in which animals are exposed to various stressors in an intermittent and random fashion, including physical restraint, footshock, alterations in the structure of the nest, etc.). Males and females that were subjected to PEE exhibited alterations in body weight, a blunted CORT response to chronic variable stress, and a reduction of testosterone (a hormone that regulates CORT levels), compared with pair-fed controls (Uban, Comeau, Ellis, Galea, & Weinberg, 2013). Altogether, these studies suggest that PEE can enhance the offspring's sensitivity to stress. This greater responsivity to aversive events may be a mechanism that underlies greater ethanol intake after PEE.

Very few studies, however, have analyzed stress-induced ethanol drinking after PEE. In early work, Nelson, Lewis, Liebeskind, Branch, and Taylor (1983) found similar ethanol intake at PD100 in rats that were exposed to ethanol throughout pregnancy and in controls, but chronic daily footshock increased ethanol intake in PEE rats only. In a more recent study (Biggio et al., 2018), offspring of dams that were subjected to ethanol intubation (1.0 g/kg/day) on GD17e20 were reared under standard conditions or exposed to daily episodes of maternal separation (i.e., a stressor that is known to increase ethanol drinking, possibly by inducing a depressive-like state) (Huot, Thrivikraman, Meaney, & Plotsky, 2001). The combination of both events, but neither PEE nor maternal separation alone, blunted baseline plasma levels of allopregnanolone, increased CORT levels after footshock exposure, and reduced the time spent on the open arms of the elevated plus maze (Biggio et al., 2018). Despite these intriguing results that suggested greater sensitivity to stress after PEE, ethanol drinking was fairly similar across groups. The present study analyzed ethanol intake after protracted prenatal and early postnatal ethanol exposure and tested whether aversive, stressful stimulation during the sensitive period of adolescence modulates the effects of PEE on ethanol drinking. Adolescents may be more sensitive to stress (Stone & Quartermain, 1997) and ethanol-stress interactions (Varlinskaya & Spear, 2012) compared with adults. Illustrating this point, we recently found that restraint stress (five 2-h sessions) significantly increased ethanol drinking a few days later in 24-h access, intermittent twobottle choice sessions in adolescent rats but significantly suppressed ethanol intake and preference in adult counterparts (WilleBille, Ferreyra, et al., 2017). Moreover, a seminal study found that exposure to adverse or stressful events during adolescence unmasked the facilitative effect of early-onset ethanol use on subsequent alcohol-related problems (Dawson, Grant, & Li, 2007). Little is known, however, about the interactive effects of stress and in utero ethanol exposure on the predisposition to ethanol intake. We replicated the lengthy prenatal and postnatal ethanol exposure of Contreras et al. (2017). At weaning (PD21), the offspring were transferred from their maternal cages to standard housing (i.e., pair housing) or isolated housing. From PD56 to PD84 (i.e., >1 month after the onset of isolation), the animals were tested for ethanol intake in 24-h access, intermittent two-bottle choice sessions that were conducted over 4 weeks. We hypothesized that PEE would enhance the facilitative effect of isolation housing on ethanol intake. Isolation was chosen based on previous reports (Butler, Karkhanis, Jones, & Weiner, 2016; Lopez, Doremus-Fitzwater, & Becker, 2011) indicating that adolescent rats may be sensitive to the effects of social isolation. During adolescence, the relevance of the social bond with the dam is progressively transferred to peers (Varlinskaya & Spear, 2015). Material and methods Experimental design and subjects A 2 (PEE: ethanol [EtOH] vs. control treatment [CTRL])  2 (housing condition after weaning: isolation vs. standard) factorial design was employed. The isolated housing groups had 13 and 14 animals each (CTRL and EtOH treatments, respectively). The standard housing groups had 12 and 13 animals each (CTRL and EtOH treatments, respectively). Subjects, housing, and control of litter effects Fifty-two outbred Sprague-Dawley rats were used (all male [27 EtOH, 25 CTRL]). They were born and reared in a temperature-


ndez et al. / Alcohol 75 (2019) 39e46 M.S. Ferna


dicas controlled vivarium in the Departamento de Ciencias Biome
lica del Norte, Coquimbo, (Facultad de Medicina, Universidad Cato Chile) and were derived from 14 litters (seven from each developmental exposure condition [EtOH, CTRL]). There were litters in which more than one male of each litter was assigned to a given cell of the experimental design. In those cases, we collapsed (i.e., averaged) the means of the siblings so that each litter only provided two values or data points to the dataset (i.e., one data point to the isolated housing group, one data point to the standard housing group). This helped prevent litter effects (Zorrilla, 1997). The animals were maintained under a 12-h/12-h light/dark cycle (lights on at 7:00 a.m.). All of the procedures were approved by the Institutional Animal Care and Use Committee of the Departamento
dicas and complied with the Declaration of de Ciencias Biome Helsinki, and Guide for the Care and Use of Laboratory Animals as promulgated by the National Institutes of Health and European Union. Maternal ethanol exposure and housing conditions after postnatal day 21 The maternal ethanol exposure protocol closely followed the procedures of Contreras et al. (2017). Briefly, time-mated pregnant dams that were housed one per cage in standard maternity cages (46 cm length  26 cm width  21 cm height) were given 10% ethanol (or its vehicle) both sweetened with sucralose as the sole fluid to drink from GD0, throughout pregnancy, until PD7. Specifically, the dams in the EtOH condition were given 22-h access per day to a bottle that contained 10% ethanol (v/v) that was mixed in tap water and sucralose (64 mg/L). To minimize the possibility of dehydration, the remaining 2 h of each day the EtOH dams had access to a bottle of tap water (20 mL, unsweetened). Throughout the day, CTRL dams were given only tap water mixed in sucralose (64 mg/L). Food chow was freely available at all times, and ethanol, water, and food consumption was recorded daily. Sucralose was included in the ethanol beverage to facilitate the acquisition and maintenance of ethanol self-administration. Compared with other sweeteners, sucralose lacks any nutritional value, has low absorption, and it is a very safe non-caloric sugar alternative (Magnuson, Roberts, & Nestmann, 2017). On PD21 (the day of weaning in most rat breeding procedures), the male offspring were housed individually (isolated housing group) in cages that measured 30 cm length  19.5 cm width  14 cm height or were housed in pairs in standard cages (standard housing group) that measured 46 cm length  26 cm width  21 cm height. Unless otherwise specified (e.g., during the ethanol intake test sessions), these conditions were maintained until termination of the experimental procedures. The female offspring were assigned to pilot, unpublished, studies. Intermittent ethanol intake assessment From PD56 to PD84, the offspring of CTRL and EtOH dams underwent an intermittent ethanol intake protocol (three weekly sessions on Mondays, Wednesdays, and Fridays; 24-h duration each). This was a variant of a standardized ethanol intake protocol that is used in our laboratory to detect the modulatory role of different vulnerability factors in ethanol consumption (e.g., re
ndez, & Pautassi, 2016). Briefly, straint stress; Acevedo, Fabio, Ferna on each ethanol intake session, the cages of the rats assigned to the isolated housing group were equipped with two bottles. One bottle contained an ethanol solution, and the other bottle contained vehicle. Rats in the standard housing group were individually housed in half of their home cage, separated from their conspecific by an opaque Plexiglas® divider. This procedure allowed the rats to

41

smell, but not touch, each other. Special lids allowed equipping each half of the cage with two bottles. Both groups (i.e., isolated or standard housing) were exposed to the concentrations of ethanol. During the first test week (PD56, PD58, and PD60), one bottle contained 5% ethanol (Porta Hnos, Cordoba, Cordoba, Argentina) that was mixed with 1% sucrose (Parker Davis; Charlotte, North Carolina, USA), and the other bottle contained 1% sucrose. During the second week (PD63, PD65, and PD67), one bottle contained 5% ethanol (mixed with 0.5% sucrose), and the other bottle contained 0.5% sucrose. During the third week (PD70, PD72, and PD74) and fourth week (PD77, PD79, and PD81), one of the bottles contained 5% ethanol that was mixed with tap water, and the other bottle contained only tap water. We preferred to test intake and preference of 5% ethanol, instead of higher concentrations, because this concentration matches the ethanol content of beverages typically consumed by adolescents and young adults. In the study by Pinsky, Zaleski, Laranjeira, and Caetano (2010), more than half of the alcohol drunk by adolescents was derived from beer (featuring 3e8% ethanol), and in another study beer was consumed by two-thirds of the young adults exhibiting binge drinking (Naimi, Brewer, Miller, Okoro, & Mehrotra, 2007). Moreover, our published (Ponce, Pautassi, Spear, & Molina, 2004) and unpublished studies indicate that uninitiated adolescent rats ingest very little uncontaminated ethanol at concentrations 6%, let alone when exposed to concentrations 20%. We did report significant ethanol intake at adolescence when ethanol is mixed with sucrose (Wille-Bille et al., 2017) or when ethanol is provided after substantial water and food deprivation (Ruiz, Calliari, & Pautassi, 2018). In the present study, we preferred to avoid the confounding effects associated with dehydration. The bottles were weighed before and after home intake sessions with an accuracy of 0.1 g. For each intake session, leakage was accounted for by placing a bottle of ethanol and a bottle of vehicle in an empty box and using post-pre liquid subtraction to correct the consumption values for the tested rats. The following variables were recorded in each session: ingestion of ethanol (g/kg; [ethanol intake (mL)  concentration of ethanol  specific weight of ethanol]/[weight of the animal (g)/1000]), percentage of ethanol preference ([ethanol intake (mL)/overall fluid intake (mL)]  100), and overall liquid consumption per 100 g of body weight ([total consumption of liquids (mL)  100 g]/body weight). Between the sessions, the animals were returned to the housing conditions of their respective groups. Data analysis The following variables were measured daily in 12 litters (six EtOH litters, six CTRL litters; data from two litters were, however, lost because of technical problems): maternal ethanol consumption (g/k/day; only for EtOH dams, average across pregnancy or nursing days), maternal food consumption (g/k/day; average during pregnancy or during nursing days), maternal fluid consumption (mL/ day; average across days), maternal consumption of additional water provided (mL/day; average across days only in EtOH dams), average maternal weight (g) during pregnancy or during breastfeeding, offspring weight (g) on PD1 and PD8, and number of pups in the litter. Significant differences in each of these variables were assessed between EtOH and CTRL litters using Student's t test. Body weight (g) that was recorded before each intake session, overall fluid consumption (mL/100 g), and absolute (g/kg) and percent (%) preference for ethanol in the offspring during the intermittent ethanol intake assessment on PD56e84 were assessed using separate repeated-measures analysis of variance (ANOVA), with prenatal ethanol exposure (EtOH, CTRL) and housing

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conditions (standard, isolated) in adolescence as between-group factors and day of ethanol intake assessment (Days 1e12) as the repeated measure. These analyses were conducted on the dataset that contained average scores for pairs of siblings that were subjected to the same housing condition during adolescence. This helped prevent the confounding effect of litter over-representation and resulted in a dataset with 28 data points (i.e., seven CTRLisolated housing, seven CTRL-standard housing, seven EtOHisolated housing, and seven EtOH-standard housing). The locus of significant main effects and significant interactions was analyzed using Fisher's Least Significant Difference post hoc test or planned comparisons. The partial eta squared (h2p) is presented as a measure of the effect size in the ANOVAs, and the a level was set at p  0.05. The post hoc tests were performed for significant effects or significant interactions that involved betweensubjects factors. Planned comparisons were used to analyze significant main effects or significant interactions that involved between-by-within factors. The rationale for this distinction was that there is no unambiguous choice of error terms for post hoc comparisons in significant effects that comprise within-subjects factors. Under these conditions, planned comparisons provide an adequate compromise between sensitivity and conservativeness (Winer, Brown, & Michels, 1991). Descriptive data are presented as mean ± SEM. Results Table 1 shows the measures recorded in EtOH and CTRL dams and litters. EtOH dams drank pharmacologically relevant concentrations of ethanol (6e8 g/kg/22 h) and, compared with CTRL dams, ate as much food during pregnancy but consumed significantly less food during nursing (t10 ¼ 2.88, p ¼ 0.02) and drank significantly less total fluid (total mL consumed, with or without ethanol) across days (t10 ¼ 6.00, p ¼ 0.001). Average maternal body weight was similar across groups during pregnancy, but EtOH dams had a significantly lower body weight during nursing (t10 ¼ 2.47, p ¼ 0.03). Litter size and offspring weight on PD1 and PD8 did not differ between conditions. As shown in Fig.1, isolation housing caused a three-fold increase in absolute and percent ethanol intake across days, a phenomenon that appeared to be exacerbated by PEE. The ANOVAs revealed significant main effects of Isolation stress and Day of assessment (g/kg: F [1,24] ¼ 77.31, h2p ¼ 0.76, p ¼ 0.001, and F[11,264] ¼ 7.78, h2p ¼ 0.25, p ¼ 0.001, respectively; percent preference: F[1,24] ¼ 46.92, h2p ¼ 0.66, p ¼ 0.001, and F[11,264] ¼ 3.33, h2p ¼ 0.12, p ¼ 0.001, respectively). The interaction between Day of assessment and Isolation stress was significant (g/kg: F[11,264] ¼ 7.90, h2p ¼ 0.25,

p ¼ 0.001; percent preference: F[11,264] ¼ 6.37, h2p ¼ 0.21, p ¼ 0.001) and the planned comparisons revealed that isolated rats drank, regardless of EtOH or CTRL treatments, significantly more ethanol compared with standard-housed rats in all (p < 0.05) but test sessions 1e3 and preferred more ethanol in all (p < 0.05) but sessions 1 to 4. The Isolation stress  PEE interaction was also significant (g/kg: F[1,24] ¼ 7.50, h2p ¼ 0.24, p ¼ 0.01; percent preference: F [1,24] ¼ 11.47, h2p ¼ 0.32, p ¼ 0.002). The post hoc tests revealed similar ethanol intake (average g/kg or percent preference across days) among rats housed in standard conditions, regardless of EtOH or CTRL treatment (p > 0.05). In contrast, among rats housed under social isolation, the post hoc tests revealed that absolute or percent ethanol intake was significantly greater (p < 0.05) in rats that were subjected to PEE (EtOH group) compared with CTRL counterparts. Ethanol intake (g/kg or percent preference) was significantly greater in PEE-isolated housing animals than in PEE-standard housing animals (p < 0.05) and significantly greater in CTRLisolated housing animals than in CTRL-standard housing animals (p < 0.05). Fig. 2 depicts the mean ethanol intake across testing days (i.e., the average of the intake scores e g/kg and % preference e registered in each ethanol intake session) and illustrates with asterisks or similar signs the significant differences yielded by the Isolation stress  PEE interaction and the subsequent post hoc tests. To further dissect patterns of ethanol intake in EtOH vs. CTRL rats, we conducted separate Isolation stress  Day of assessment ANOVAs, one for each PEE condition (i.e., one for the EtOH group and one for the CTRL group). The aim was to analyze the temporal dynamics of the effect of isolation stress on ethanol intake and further confirm that isolation stress exerted differential effects as a function of PEE. The ANOVA of g/kg ethanol ingested revealed significant main effects of Stress treatment and Day of assessment (EtOH group: F[1,12] ¼ 94.86, h2p ¼ 0.88, p ¼ 0.001, and F [11,132] ¼ 3.99, h2p ¼ 0.25, p ¼ 0.001, respectively; CTRL group: F [1,12] ¼ 14.10, h2p ¼ 0.52, p ¼ 0.003, and F[11,132] ¼ 4.05, h2p ¼ 0.25, p ¼ 0.001, respectively) and a significant Stress treatment  Day of assessment interaction (EtOH group: F [11,132] ¼ 3.99, h2p ¼ 0.25, p ¼ 0.001; CTRL group: F[11,132] ¼ 4.29, h2p ¼ 0.26, p ¼ 0.001). The planned comparisons indicated that the facilitative effect of isolation stress on ethanol intake in the EtOH group emerged at the first free-choice session and lasted throughout the end of testing (p < 0.05), with the exception (p > 0.05) of session 3 (Fig. 1B). In sharp contrast, throughout most of the first 2 weeks of testing (i.e., sessions 1e5; Fig. 1A) CTRL animals that were subjected to isolation stress exhibited ethanol intake that was similar (p > 0.05) to that of CTRL animals housed in pairs in standard cages. A significant effect of isolation stress (p < 0.05) was observed in CTRL animals in sessions 6e12.

Table 1 Parameters measured in ethanol (EtOH) and control (CTRL) dams and their litters.

Maternal ethanol consumption (g ethanol/kg/day) Maternal food consumption pregnancy (g/kg/day) Maternal food consumption nursing (g/kg/day) Maternal liquid consumption (mL/day) Maternal consumption of the daily liquid complement (unsweetened tap water, mL/day) Average of maternal weight (g) during pregnancy Average of maternal weight during nursing (g) Offspring weight e postnatal 1 day (g) Offspring weight e postnatal 7e8 day (g) Litter size

Maternal treatment EtOH

Maternal treatment CTRL

6.06 ± 0.07a 8.17 ± 0.81b 76.09 ± 6.6 99.54 ± 4.7 28.18 ± 2.833 17.23 ± 0.61 317.4 ± 13.42 296.3 ± 10.15 7.05 ± 0.87 13.74 ± 0.92 13.67 ± 0.61

NA 78.10 ± 1.7 121.4 ± 6 47.11 ± 1.38 NA 333.4 ± 12.5 341.6 ± 15.3 6.7 ± 0.15 14.47 ± 0.54 12.67 ± 0.55

p

ns * *** ns * ns ns ns

Both maternal treatments had sucralose (64 mg/L); p ¼ statistical probability, *p < 0.05, ***p < 0.001, as indicated by Student's t test. Data are presented as mean ± SEM, n ¼ 6. NA: not applicable. a During pregnancy. b During nursing.


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Fig. 1. Ethanol intake (g/kg) (A, B) and percent preference of ethanol consumption (C, D) in male rats as a function of maternal ethanol exposure throughout gestation until PD7. The dams in the ethanol condition were given 22-h access per day to a bottle that contained 10% ethanol (v/v) that was mixed in tap water and sucralose (64 mg/L). Control dams were given only tap water and sucralose. The housing conditions that were experienced since weaning consisted of standard control housing and social isolation housing (standard and isolated groups, respectively). The intake sessions were conducted between PD56 and PD81. Two-bottle intake sessions (5% ethanol vs. vehicle) were conducted on Monday, Wednesday, and Friday (22-h session length) for 4 weeks. *p < 0.05, significant difference between standard-housing and isolated-housing rats on a given day of testing among the ethanol and control groups. The data are expressed as mean ± SEM.

Similar to the ANOVA of absolute ethanol intake scores, the ANOVA of percent ethanol preference revealed a significant main effect of Day of assessment in the EtOH and CTRL groups and a significant effect of Stress treatment only in the EtOH group (EtOH

group: F[11,132] ¼ 4.21, h2p ¼ 0.26, p ¼ 0.001, and F[1,12] ¼ 81.17, h2p ¼ 0.87, p ¼ 0.001, respectively; CTRL group: F[11,132] ¼ 3.38, h2p ¼ 0.13, p ¼ 0.001) and a significant Stress treatment  Day of assessment interaction (EtOH group: F[11,132] ¼ 4.21, h2p ¼ 0.26,

Fig. 2. Same data as in Fig. 1, averaged across day of ethanol intake testing. Ethanol intake (g/kg) (A) and percent preference of ethanol consumption (B) in male rats as a function of maternal ethanol exposure throughout gestation until PD7. The dams in the ethanol condition were given 2-h access per day to a bottle that contained 10% ethanol (v/v) that was mixed in tap water and sucralose (64 mg/L). Control dams were given only tap water and sucralose. The housing conditions that were experienced since weaning consisted of standard control housing and social isolation housing (standard and isolated groups, respectively). *p < 0.05, #p < 0.05, significantly greater ethanol intake in ethanol-isolated housing rats than in ethanol-standard housing and control-isolated housing rats, respectively; &p < 0.05, significantly greater ethanol intake in control-isolated housing rats than in control-standard housing rats. The data are expressed as mean ± SEM.


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p ¼ 0.001; CTRL group: F[11,132] ¼ 3.38, h2p ¼ 0.21, p ¼ 0.001). In the CTRL group (Fig. 1C), the planned comparisons indicated that isolation stress increased ethanol intake (Fig. 2B) only in sessions 7, 10, 11, and 12 (p < 0.05). In the EtOH group (Fig. 1D), isolation stress induced greater ethanol intake compared with the standardhoused group in all free-choice sessions (p < 0.05) except sessions 2 and 3 (p > 0.05) (Fig. 2C). The ANOVA of body weight that was recorded before each intake session revealed significant main effects of Day of assessment and PEE (F[11,264] ¼ 2.23, h2p ¼ 0.09, p ¼ 0.01, and F [1,24] ¼ 7.11, h2p ¼ 0.23, p ¼ 0.01, respectively; Table 2). The EtOH rats exhibited significantly lower body weights than CTRL rats, and body weights gradually increased across days. The ANOVA of overall fluid consumption (mL/100 g) in the offspring during the ethanol intake assessments revealed significant main effects of Isolation stress and Day of assessment (F[1,24] ¼ 44.65, h2p ¼ 0.65, p ¼ 0.001, and F[11,264] ¼ 30.98, h2p ¼ 0.56, p ¼ 0.001, respectively) and a significant Isolation stress  Day of assessment interaction (F[11,264] ¼ 4.71, h2p ¼ 0.16, p ¼ 0.001). The planned comparisons revealed that isolated rats drank significantly more fluid than standard-housed rats in sessions 1e9 (p < 0.05) but not in sessions 10e12 (p > 0.05). Isolated, but not standard-housed, rats exhibited a decrease in overall fluid intake in session 7 (p < 0.05) compared with the previous sessions. These results were unaffected by PEE (Table 2). Discussion Consistent with previous studies (Kutcher, Egorov, & Chernikova, 2016; Lopez et al., 2011), prolonged isolation housing during adolescence increased subsequent free-choice ethanol drinking in young adulthood. Across sessions, isolation induced a three-fold increase in absolute and percent ethanol drinking compared with standard-housed rats. The effect sizes were 0.65e0.70, which were above the cut-off point for a large effect size (i.e., >0.30) (Cohen, 1988). The main novel result of the present study was that the facilitative effect of isolation on absolute and percent ethanol intake was significantly greater in PEE rats than in their CTRL counterparts. The effect sizes of the combined influence of PEE and isolation on absolute and percent ethanol intake were at or around the threshold for a large effect size (i.e., 0.24 and 0.32, respectively). Moreover, we

employed a repeated ethanol intake testing that allowed assessing the temporal dynamics of the effect of isolation stress, within each prenatal treatment group. PEE-isolated housing rats began to exhibit an increase in ethanol consumption, compared with PEEstandard housing rats, from the very first ethanol intake test, whereas a similar effect of isolation housing was observed in CTRL rats after 2 weeks of testing, approximately. In other words, PEE appeared to unmask the effect of isolation that was otherwise latent during the first 2 weeks of testing in CTRL-isolated housing rats. These results resemble those of Dawson et al. (2007), who studied a large sample of drinkers (n ¼ 26,946) and found that the association between stress and ethanol consumption achieved significance only in individuals who had the first drink at 14 years of age or younger. Altogether, the present results and these previous findings suggest that early ethanol exposure (e.g., prenatally or during adolescence) renders individuals vulnerable to stress or decreases their ability to cope with stress. Infants whose mothers ingested alcohol from pregnancy until recognition of the pregnancy exhibited increases in heart rate and cortisol levels and negative affect when emotionally challenged (Haley et al., 2006). The reliability of the effect of stress in the present study may appear surprising, particularly when considering previous studies that reported disparate data with regard to the effects of aversive stimulation on ethanol intake. A review by Becker et al. (2011) indicated that no consensus has been reached with regard to the effects of stress-ethanol interactions on ethanol intake. Some studies reported an increase in ethanol intake, whereas others reported a decrease in intake or no effect of comparable treatments. It is tempting to speculate that sex has a significant role in stressethanol interactions. Yet, as highlighted by Becker et al. (2011), there are still very few studies that explicitly compared both sexes, and those available have provided mixed evidence. Some stressors, however, have produced results that are more consistent. Prolonged isolation stress in rodents (Holgate, Garcia, Chatterjee, & Bartlett, 2017) usually significantly increases ethanol drinking compared with non-stressed rodents, an effect that is particularly reliable when isolation occurs during adolescence, such as in the present study (Kutcher et al., 2016; Lopez et al., 2011). This may be related to the fact that peer-to-peer interactions are more important during adolescence than during infancy or adulthood (Varlinskaya & Spear, 2015), and this can be exacerbated when

Table 2 Overall fluid intake (mL/100 g of body weight) and body weight (g), expressed as a function of prenatal ethanol exposure (control or ethanol treatment) and housing condition after weaning (isolated housing vs. standard housing) during ethanol intake sessions (112) conducted from DP56 to DP81 in male rats. Ethanol intake session

Developmental Ethanol Exposure Control

1 2 3 4 5 6 7 8 9 10 11 12

Ethanol

Standard housing

Isolated housing

Overall fluid consumption (mL/100 g)

Body weight (g)

Overall fluid consumption (mL/100 g)

9.54 ± 0.95 14.77 ± 1.58 13.71 ± 0.79 11.66 ± 1.18 13.22 ± 1.18 9.03 ± 0.93 8.34 ± 0.34 9.76 ± 0.47 9.42 ± 0.53 8.79 ± 0.41 11.94 ± 0.73 11.94 ± 2.63

296.60 ± 8.70 306.71 ± 8.74 320.05 ± 8.83 328.88 ± 8.25 345.52 ± 9.88 355.52 ± 10.73 373.64 ± 11.92 386.79 ± 10.67 393 ± 11.22 403.98 ± 11.75 416.69 ± 11.74 422.45 ± 11.80

20.30 25.70 19.42 21.29 20.10 24.30 11.19 12.51 12.77 11.18 11.44 11.50

± ± ± ± ± ± ± ± ± ± ± ±

2.62 2.67 2.19 2.42 2.56 2.37 1.60 0.83 0.68 0.99 0.73 1.09

Standard housing

Isolated housing

Body weight (g)

Overall fluid consumption (mL/100 g)

Body weight (g)

Overall fluid consumption (mL/100 g)

Body weight (g)

310.38 ± 7.23 318.38 ± 7.72 325 ± 8.81 342.24 ± 11.14 351.48 ± 7.54 364.95 ± 9.39 387.62 ± 9.65 396.05 ± 11.13 401.14 ± 9.86 405.14 ± 12.26 414.71 ± 10.07 422.38 ± 11.48

14.72 ± 1.44 16.89 ± 2.34 17.37 ± 1.96 15.16 ± 1.61 11.88 ± 1.47 12.54 ± 1.22 9.40 ± 0.93 9.40 ± 0.34 7.96 ± 0.45 9.67 ± 0.51 9.52 ± 0.96 9.24 ± 0.78

260.86 ± 11.67 278 ± 13.06 288.29 ± 12.75 303 ± 13.56 316.52 ± 14.50 323.43 ± 13.97 351.67 ± 14.55 346.05 ± 14.42 357.95 ± 14.77 368.71 ± 14.54 379.38 ± 15.98 387.19 ± 16.97

15.82 ± 1.71 21.89 ± 1.29 22.51 ± 2.37 20.50 ± 2.13 18.91 ± 2.15 21.24 ± 1.65 12.99 ± 1.11 12.19 ± 0.87 13.49 ± 0.92 10.78 ± 1.01 10.19 ± 0.41 9.94 ± 0.41

288.48 ± 9.06 294.76 ± 10.45 303.87 ± 10.80 314.70 ± 14.74 331.56 ± 10.94 339.38 ± 14.35 359.27 ± 12.48 368.70 ± 12.34 370.20 ± 10.47 374.35 ± 9.42 384.92 ± 10.55 391.8 ± 9.54

Values express mean ± SEM. Please refer to the text for an account of the significant main effects or significant interactions found.


ndez et al. / Alcohol 75 (2019) 39e46 M.S. Ferna

isolation is imposed immediately after removing the dam at weaning. It is also possible that greater brain plasticity at a young age contributes to the protracted effects of stress during adolescence. The present results have limitations and caveats. Social isolation significantly increased overall fluid consumption during the intake sessions, and PEE significantly reduced body weights. The ethanol intake results could reflect these alterations. A dissociation was found, however, between patterns of ethanol intake and these effects. Overall fluid intake was unaffected by PEE, and PEE reduced body weights of both isolated and standard-housed animals, but only PEE-isolated housing rats exhibited increases in ethanol intake and preference. Another limitation of the present study was that we only assessed male rats because of logistic and budgetary reasons. Therefore, these findings should be generalized to female rats with caution. Previous studies from our laboratory indicated that adolescent female rats were more sensitive to stress-induced drinking than their male counterparts (Wille-Bille, Ferreyra, et al., 2017), yet other studies of adolescent stress and ethanol drinking show no increased drinking in females (Roeckner, Bowling, & Butler, 2017). Lastly, EtOH dams consumed significantly less food and had lower body weights compared with control dams. These effects, however, were only observed during nursing, a stage in which the demands of caring for pups challenge the energy resources of the dam. Moreover, despite these effects, the size of the litters and the weight of the offspring were similar on PD1 and PD8. Another important limitation is that we did not measure basal or stress-induced tone of the HPA axis, molecular markers of stress, or anxiety-like behavior in a validated model of anxiety. This lack of additional measures limits interpretation of the results. However, based on our previous studies, alterations in anxiety-like responses or redox state may underlie the greater sensitivity to stress-induced drinking in PEE rats. Contreras et al. (2017) employed the same preand postnatal ethanol exposure paradigm as in the present study and found PEE-induced alterations in catalase, superoxide dismutase, glutathione peroxidase 1, and nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2) enzymes, which are involved in redox homeostasis and alterations in N-methyl-D-aspartate receptor subunits. Moreover, the inhibition of NOX2 in the ventral tegmental area blocked an ethanol-induced conditioned preference in PEE rats only (Contreras et al., 2017). In other work, we found that PEE rats exhibited a decrease in the time spent in the open areas of the lightdark box and greater shelter-seeking in a modified version of the multivariate concentric square field (Wille-Bille et al., unpublished data), a pattern that is indicative of an anxiety-prone phenotype that is likely to facilitate ethanol intake (McCaul, Hutton, Stephens, Xu, & Wand, 2017). Yet another limitation is that we did not measure blood ethanol concentrations (BECs) in the dams. If their daily ethanol intake was distributed evenly across 22 h, then it would be possible that they never achieved pharmacologically relevant BECs. We did, however, measure g/kg and BECs in a study (Contreras et al., 2017) that employed the same protocol of developmental ethanol exposure. In Contreras et al. (2017) we found that the dams drank an average of 8.1 ± 0.4 g/kg/day of ethanol during the pre- and postnatal treatment period, which is very similar to the values of the present study. BECs were measured 12 h after commencement of the dark period and indicated that dams achieved a concentration of 62 ± 22 mg/dL (Contreras et al., 2017). Despite these limitations, the present results suggest that PEE renders the individual sensitive to the facilitative effect of stress on ethanol intake. These results support the hypothesis (Chotro et al., 2007) that PEE or, more broadly, early developmental ethanol exposure (Dawson et al., 2007) exerts long-term alterations on ethanol intake patterns by increasing the sensitivity to stress or to stress-ethanol interactions.

45

Declaration of interest None. Acknowledgments and funding sources This work was a collaborative project between the Facultad de
lica del Norte (Chile), and Instituto M. y Medicina, Universidad Cato M. Ferreyra (Argentina) and was supported by PICT 2015-0325 to RMP, FONDECYT 1140855 to PHS, and a PROLAB cooperation grant from IBRO that was awarded jointly to both senior authors (PHS and RMP). The authors would like to thank Sofia Vargas-Roberts and Daniel Rojas for technical assistance and Cristopher Collao,
pez, Carolina Toledo, and Catalina ValVanessa Flores, Romina Lo
lica del ladares (students of the Medical School, Universidad Cato Norte). References
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