A novel mouse model for vulnerability to alcohol dependence induced by early-life adversity

Childhood adversity increases vulnerability to alcohol use disorders and preclinical models are needed to investigate the underlying neurobiological mechanisms. The present study modeled early-life adversity by rearing male and female C57BL/6J mouse pups in a limited bedding and nesting (LBN) environment, which induces erratic maternal care. As adults, mice were given limited access to two-bottle choice (2BC) alcohol drinking, combined or not with chronic intermittent ethanol (CIE) vapor inhalation to induce alcohol dependence. We tested the hypothesis that LBN rearing might exacerbate or facilitate the emergence of the motivational and affective effects of CIE. Consistent with our hypothesis, although LBN-reared males consumed the same baseline levels of alcohol as controls, they escalated their ethanol intake at an earlier stage of CIE exposure, i.e., after 4 rounds vs. 5 rounds for controls. In contrast, females were insensitive to both LBN rearing and CIE exposure. Males were further subjected to a behavioral test battery. Withdrawal from CIE-2BC increased digging activity and lowered mechanical nociceptive thresholds regardless of early-life conditions. On the other hand, LBN-reared CIE-2BC males showed reduced open arm exploration in the elevated plus maze and increased immobility in the tail suspension test compared to alcohol-naïve counterparts, while no group differences were detected among control-reared males. Finally, LBN rearing and alcohol exposure did not affect grooming in response to a sucrose spray (splash test), novel object recognition, or corticosterone levels. In summary, the LBN experience accelerates the transition from moderate to excessive alcohol drinking and produces additional indices of affective dysfunction during alcohol withdrawal in C57BL/6J male mice. Virgin 8-week-old female (n = 16) and male (n = 6) C57BL/6J mice (stock #000664) were obtained from The Jackson Laboratory (Sacramento, CA) and housed in an uncrowded, quiet animal facility room on a 12-h light/dark cycle with free access to lab chow and water. Beginning on postnatal day (P)75, 2–3 females were paired with each male for breeding. Females were examined daily for evidence of a vaginal plug (confirmation of successful mating, considered to be embryonic day [E]0). Pregnant females were separated on E17, prior to parturition. Dams were checked for parturition every 12 h, and the day of birth was considered P0. All procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of University of California – Irvine (UCI) and The Scripps Research Institute (TSRI). Ethanol (200 proof) was obtained from PHARMCO-AAPER (Brookfield, CT). 15% (v:v) ethanol was prepared by diluting ethanol in reverse osmosis-purified water. Pyrazole was obtained from Sigma (St Louis, MO). Pyrazole was dissolved in either saline or 20% (v:v) ethanol in saline. Fresh solutions were prepared weekly. The experimental timeline is summarized in Fig. 1A. C57BL/6J male and female offspring were reared under LBN (n = 22 males and 11 females) or Control (n = 24 males and 15 females) conditions from P2 to P9 at UCI. All mice were transferred to TSRI when they were 3.5–5 weeks old. Six weeks later, they were moved to reverse light cycle rooms (at which point they experienced an 8-h phase advance of the light cycle), habituated for 6 days (males) or 13 days (females), and single-housed 3 days before testing started. C57BL/6J mice need 5.7 ± 0.7 days to re-entrain their circadian rhythm following an 8-h advance of the light-dark cycle, so males had theoretically recovered from the shift by the time testing started (Legates et al., 2009). The LBN paradigm has been shown to produce sex-dependent consequences in several behaviors and brain functions (reviewed in Walker et al., 2017). Based on this knowledge, we decided a priori to analyze data from males and females separately and optimize our experimental design to detect an interaction between early-life history and vapor exposure within each sex. For logistic reasons, it was impossible to test 72 mice in parallel. Accordingly, the male and female cohorts were staggered by one week. Mice remained single-housed in Sani-Chips bedding (Envigo, Placentia, CA) with ad libitum access to reverse osmosis-purified water and sterilized food chow (Teklad LM-485, Envigo) throughout the duration of the experiment. All behavioral testing was conducted during the dark phase under red light illumination (light offset at 10:00 a.m. for males, 9:00 a.m. for females). A. Experimental timeline. C57BL/6J male and female pups were reared under limited bedding and nesting (LBN) or control conditions from postnatal days (P) 2–9. Once mice reached adulthood, they were subjected to limited-access two-bottle choice (2BC) ethanol drinking (grey boxes). Once ethanol intake stabilized, some of the mice were exposed to chronic intermittent ethanol (CIE) vapor inhalation (blue boxes). 2BC sessions were alternated with vapor inhalation over the course of 14–16 weeks. After their last week of 2BC, males were subjected to the elevated plus maze (EPM), digging (Dig), splash (Groom), and tail suspension (TST) tests. They were then exposed to an additional week of CIE and tested in the tail pressure test (TPT) 32 h into withdrawal. After a final week of CIE, they were tested for novel object recognition (NOR) and blood was collected for corticosterone measurement (Cort). B–C. Body weights in males (B) and females (C) at the end of the early-life adversity (P10), at weaning (P21) and at the beginning of alcohol drinking (P70-91). * or **, effect of LBN, p < 0.05 or p < 0.01. Mice were first subjected to eleven sessions of two-bottle choice (2BC) alcohol drinking to stabilize their ethanol intake. Males from each early-life condition were then split into 3 subgroups of equivalent baseline intake: 1. Air-water mice (Control, n = 8; LBN, n = 6), which had access to two bottles of water during 2BC sessions from that point onward and inhaled air only, 2. Air-2BC mice (Control, n = 8; LBN, n = 8), which had access to ethanol and water during 2BC sessions and inhaled air only, and 3. CIE-2BC mice (Control, n = 8; LBN, n = 8), which had access to ethanol and water during 2BC sessions and were exposed to chronic intermittent ethanol (CIE) vapor inhalation. Due to a smaller sample size, females were split in only 2 subgroups: Air-2BC (Control, n = 7; LBN, n = 5) and CIE-2BC (Control, n = 8; LBN, n = 6). Each experimental subgroup contained up to 4 mice from the same litter. 2BC sessions and vapor exposure were then alternated in two phases. During the priming phase, 2BC sessions were followed by one night (16 h) of ethanol vapor (or air) inhalation (5:00 p.m.-9:00 a.m.), for a total of 6 times, with at least 48 h between the end of vapor exposure and the subsequent 2BC session. This priming phase was meant to enable the detection of group differences at an early stage of CIE exposure. Weeks of CIE (or air) inhalation (4 × 16-h intoxication/8-h withdrawal, Mon-Fri) were then alternated with weeks of 2BC (Mon-Fri) for a total of 5 (males) or 6 (females) rounds, as described previously (Becker and Lopez, 2004; Kreifeldt et al., 2013). After their last week of 2BC, males were subjected to the elevated plus maze, digging, splash, and tail suspension tests (10–11, 13, 17 and 19 days after last vapor exposure, respectively). They were then exposed to an additional week of CIE and tested in the tail pressure assay 32 h into withdrawal. They were exposed to a final week of CIE and tested for novel object recognition (habituation to arena, training, and testing on withdrawal days 3, 5 and 6, respectively) and blood was collected for corticosterone measurement on withdrawal day 7. One LBN male died 2 days into withdrawal from this last vapor exposure. The small size of the Control and LBN female groups did not allow for the constitution of Air-water subgroups. Furthermore, one LBN Air-2BC female and one LBN CIE-2BC female died during the course of the alcohol drinking experiment. Accordingly, the experimental design would have been suboptimal and underpowered for the analysis of affective, nociceptive, and cognitive phenotypes during withdrawal from CIE-2BC in females. Early-life adversity was induced P2–P9 via an impoverished environment with limited bedding and nesting (referred to as LBN), as described previously (Rice et al., 2008). Briefly, on the morning of P2, litters were adjusted to no more than 8 pups, yielding litter sizes of 5–8. Control dams and litters (n = 7) were placed in cages with standard amounts of corn husk bedding (~650 mL) and one square piece of cotton-like nesting material measuring 5 cm × 5 cm. This material is shredded by the dam to create a nest area for her pups. LBN dams and litters (n = 6) were placed in cages fitted with a fine-gauge plastic-coated aluminum mesh platform (mesh dimensions 0.4 × 0.9 cm, catalog no. 57398; McNichols Co., Tampa, FL) sitting ~2.5 cm above the cage floor. Bedding was reduced to only sparsely cover the cage floor (~60 mL), and one-half of a square of nesting material was provided. Both groups were completely undisturbed until the morning of P10, at which point all cages were changed to standard cages with ample bedding and nesting material. At P21, pups were weaned and housed with same-sex littermates in groups of 3–5. Body weights were recorded at P10 and P21. Voluntary ethanol consumption was assessed in 2-h sessions during which mice had access to a bottle of water and a bottle of 15% (v:v) ethanol in their home cage, as described previously (Becker and Lopez, 2004; Kreifeldt et al., 2013). Sessions started at the beginning of the dark phase and were conducted Mon-Fri. The position of the ethanol and water bottles was alternated between sessions to control for side preference. Ethanol intake was determined by weighing bottles before and after the session, subtracting the weight lost in bottles placed in an empty cage (to control for spill/evaporation) and dividing by the mouse body weight (measured weekly). Ethanol vapor exposure was conducted as described previously (Becker and Lopez, 2004; Kreifeldt et al., 2013). The inhalation chambers were made of sealed standard plastic mouse cages. An electronic metering pump (Iwaki EZB11D1-PC) dripped 95% ethanol into a flask placed on a warming tray at a temperature of 50 °C. Drip rate was adjusted to achieve target BECs of 150–250 mg/dL. An air pump (Hakko HK-80L) conveyed vaporized ethanol from the flask to each individual chamber. The air flow was set at a rate of 15 L/min for each pair of chambers. Each chamber was diagonally divided by a mesh partition to provide single housing for two mice. Mice received a loading injection of ethanol (1.5 g/kg) and pyrazole (68 mg/kg) in saline, intraperitoneally administered in a volume of 0.1 mL/10 g body weight, before each 16-h ethanol vapor inhalation session. Pyrazole, an alcohol dehydrogenase inhibitor, served to maintain stable BECs during the 16-h period. Air-water and Air-2BC mice also received pyrazole. Blood was sampled from the caudal vein once a week in each CIE-2BC mouse, at the end of a 16-h intoxication session. The tip of the tail was nicked with a scalpel blade, blood was collected with a heparinized capillary tube and centrifuged at 13,000 g for 10 min. BECs were measured by gas chromatography and flame ionization detection (Agilent 7820A). The EPM apparatus consisted of a 5 cm × 5 cm central square connected to two opposite open arms and two opposite closed arms. Each arm was 30-cm long and 5-cm wide. The closed arms had 15-cm high walls while the open arms had 3-mm high ledges. The runway was placed on top of a 30-cm high stand. The runway floors were made of matte grey acrylic, all other surfaces were made of clear acrylic. The mouse was placed in the central square of the apparatus, facing a closed arm, and allowed to explore freely for 5 min. The apparatus was wiped with 70% ethanol in between mice. The test was recorded by a camera mounted above the EPM and connected to a computer. The distance traveled, number of entries, and time spent in each area of the EPM were calculated by the ANY-maze program (Stoelting Co., Wood Dale, IL). The total distance traveled was used as an index of locomotor activity and the time spent in the open arms was used as an index of anxiety-like behavior, as previously described (Komada et al., 2008; Rodgers and Dalvi, 1997). The apparatus design (open arms with ledges and closed arms with transparent walls) was meant to encourage exploration of the open arms and facilitate the detection of an anxiogenic-like effect of alcohol withdrawal (Dere et al., 2002; Horii and Kawaguchi, 2015). Digging activity was assessed according to Deacon (2006). The mouse was placed in the middle of a standard acrylic mouse cage (with no lid) filled with a 5-cm depth of Sani-Chip bedding (Envigo). The latency to dig, number of digging bouts and duration of digging were measured using stopwatches and a tally counter for a total duration of 3 min. Bedding was changed between each mouse. Testing was conducted under red light. Digging is a spontaneous, species-typical behavior performed by wild mice foraging for buried food in their natural habitats and exhibited by laboratory mice placed on a thick layer of bedding substrate. Digging behavior is sensitive to multiple psychotropic drugs (see Deacon, 2006 and references therein) and we previously reported that it is robustly increased during withdrawal from CIE (Sidhu et al., 2018). The splash test was conducted in the home cage, under red light, using the method of Ducottet et al. (2004). A solution of 10% sucrose was sprayed on the dorsal coat of the mouse using a single squirt from a standard gardening spray bottle in mist position. The latency to groom, the number of grooming bouts and duration of grooming were measured using stopwatches and a tally counter for a total duration of 5 min. Grooming activity is used as an index of self-care that is degraded in mouse models of depressive-like behavior (d'Audiffret et al., 2010; Yalcin et al., 2005). This test was used to assess the coping strategy used by mice facing an acute, inescapable stress (Cryan et al., 2005; Steru et al., 1985). The tail of the mouse was inserted in a hollow cylinder (3.5-cm length, 1-cm diameter, 1 g) to prevent tail climbing as described by Can et al. (2012). The 2-cm end of a 17-cm piece of tape was adhered to the mouse tail and back to itself, leaving the distal 2–3 mm of the tail protruding out of the tape. The other end of the tape was stuck to a shelf placed 30 cm above the bench, such that the tape hung vertically. The duration of immobility of each mouse was measured for 6 min. Relative levels of immobility were interpreted in terms of active vs. passive stress-coping strategy (Anyan and Amir, 2018; Commons et al., 2017; Molendijk and de Kloet, 2015). Mechanical nociceptive thresholds were assessed by applying pressure on the tail using a digital Randall-Selitto apparatus (Harvard Apparatus, Holliston, MA), as previously described by Elhabazi et al. (2014). The mice were first habituated to enter a restrainer pouch made of woven wire (stainless steel 304L 200 mesh, Shanghai YiKai) over three days. On testing days, the mouse was gently introduced into the restrainer and the distal portion of the tail was positioned under the conic tip of the apparatus. The foot switch was then depressed to apply uniformly increasing pressure onto the tail until the first nociceptive response (struggling or squeaking) occurred. The force (in g) eliciting the nociceptive response was recorded. A cutoff force of 600 g was enforced to prevent tissue damage. The measure was repeated on the medial and proximal parts of the tail of the same mouse, with at least 30 s between each measure. The average of the three measures (distal, medial, proximal) was used for statistical analysis. Long-term object memory was assessed as described by Lueptow (2017). Testing was conducted under white light (350 lux). Mice were individually habituated during 10 min to a 50 cm × 50 cm enclosure with 38-cm high walls made of beige ABS plastic (San Diego Instruments 7001-0067) and lined with bedding. Twenty-four hours later, two identical objects (20-mL glass vial filled with water) were taped to the floor of the arena, 11 cm away from two adjacent corners and 22 cm away from each other, and the mouse was given 10 min to freely explore them. On the following day, one of the two objects was replaced with another object (medium-sized paper clip) and the mouse was again given 10 min to explore. The side on which the object was replaced with a novel object was counterbalanced across mice. The arena and objects were wiped with ethanol (70% v:v) between mice to remove odor cues. The mouse head position was video-tracked and circular areas extending 2 cm away from each object were defined in ANY-maze. The discrimination index was calculated as the ratio of time spent in the novel object circle over time spent in the familiar object circle. One Control CIE-2BC mouse was identified as an outlier using the Grubbs’ test (Grubbs, 1969) and was therefore excluded. Mice were euthanized by cervical dislocation immediately followed by decapitation, 2–5 h into the dark phase. Trunk blood was collected into EDTA-coated tubes. Plasma was separated by centrifugation at 1600 g for 10 min at 4 °C and stored at −80 °C. Corticosterone levels were measured in duplicates using the Corticosterone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, MI) according to the manufacturer's instructions. Data analysis was performed in Statistica 13.3 (TIBCO Software Inc.). Data from males and females were analyzed separately. Linear mixed-model analyses were used to evaluate litter effects (Zorrilla, 1997) and determine the effect of LBN while accounting for intralitter likeness, as per Golub and Sobin (2020). Early-life history was defined as a fixed factor and litter as a random factor nested under early-life history. Expected mean squares were estimated using the over-parameterized model and degrees of freedom were computed using Satterthwaite's method of denominator synthesis. To determine the effect of CIE and the interaction of LBN with CIE, data were analyzed by two-way analysis of variance (ANOVA) with early-life history (Control, LBN) and alcohol exposure (Air-water [when relevant], Air-2BC, CIE-2BC) as between-subject variables. Tukey posthoc tests were conducted when appropriate. Repeated measures ANOVA (RM-ANOVA, with time as within-subject variable) was used to analyze ethanol drinking acquisition and escalation. Planned comparisons were conducted using two-tailed unpaired t-tests to test our hypothesis that LBN facilitates the effect of CIE on ethanol intake and negative affect. To further examine the trajectory of ethanol intake escalation in each group, the proportion of mice whose weekly average intake exceeded 4 g/kg was subjected to Kaplan-Meier survival analysis. We selected 4 g/kg as a criterion because a consumption of 4 g/kg in 2 h yields BECs ≈150 mg/dL (Becker and Lopez, 2004; Contet et al., 2014), which corresponds to twice the BEC threshold for ethanol intoxication, as well as to the lower end of the BEC range targeted during ethanol vapor inhalation. Data are shown as mean ± s.e.m. in the graphs.