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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: Alcohol Clin Exp Res. 2021 Oct 4;45(11):2231–2245. doi: 10.1111/acer.14717

Alcohol self-administration and effects of sex in APOE targeted replacement mice before and after repeated mild traumatic brain injury using the automated IntelliCage system

Kathryn E Simmons 1, Kati L Healey 2, Qiang Li 2, Scott D Moore 2,3, Rebecca C Klein 2,3
PMCID: PMC8651057  NIHMSID: NIHMS1741432  PMID: 34585391

Abstract

Background:

There are few studies examining the association between APOE genotype and alcohol use. While outcomes associated with a history of drinking have been reported, none of these studies have examined alcohol seeking behavior. In addition, no pre-clinical studies have been conducted to examine alcohol use as a function of APOE genotype with or without traumatic brain injury.

Methods:

Male and female human APOE3 and APOE4 targeted replacement (TR) mice were used to longitudinally assess voluntary alcohol seeking using a two-bottle choice paradigm conducted within the automated IntelliCage system prior to and following repeated mild TBI (rmTBI). Following acquisition to increasing % EtOH up to 12%, a variety of drinking paradigms including extended alcohol access (EAA1 and EAA2), alcohol deprivation effect (ADE), limited access drinking in the dark (DID), and progressive ratio (PR) were used to assess alcohol seeking behavior. Additional behavioral tasks were performed to measure cognitive function and anxiety-like behavior.

Results:

All groups readily consumed increasing concentrations of EtOH (4–12%) during the acquisition phase. During the EAA1 period (12% EtOH), there was a significant genotype effect in both males and females for EtOH preference. Following a 3 week abstinence period, mice received sham or rmTBI resulting in a genotype- and sex-independent main effect of rmTBI on the recovery of righting reflex and a main effect of rmTBI on spontaneous home cage activity in females only. Reintroduction of 12% EtOH (EAA2) resulted in a significant effect genotype for alcohol preference in males with APOE4 mice displaying increased preference and motivation for alcohol compared to APOE3 mice independent of TBI while in females there was a significant genotype X TBI interaction under the ADE and DID paradigms. Finally, there was a main effect of rmTBI on increased risk-seeking behavior in both sexes, but no effect on spatial learning or cognitive flexibility.

Conclusion:

These results suggest that sex and APOE genotype play a significant role in alcohol consumption and may subsequently influence long-term recovery following traumatic brain insults.

Introduction

There is a well-established connection between traumatic brain injury (TBI) and alcohol misuse: it is estimated that over 25% of individuals with TBI become moderate to heavy drinkers post-injury (Bombardier et al., 2003, Grossbard et al., 2017). Excessive and problematic alcohol use following TBI has been associated with negative physical, cognitive and behavioral outcomes (Weil et al., 2016a). Given that chronic alcohol use is associated with numerous cognitive and behavioral deficits, understanding the etiology of post-injury alcohol seeking behavior is of major concern. Further, genetic factors likely contribute to TBI outcome, in particular the apolipoprotein E gene (APOE4) is associated with poorer long term outcomes in cognition following TBI (Yue et al., 2017, Zhou et al., 2008), and worse severity of initial injury (Smith et al., 2006). Until now, no preclinical studies have examined the interaction between the APOE4 genotype and alcohol use in the context of TBI.

The APOE4 gene is a major genetic risk factor for Alzheimer’s disease (AD) and is associated with poorer learning and memory in middle aged individuals (Flory et al., 2000). APOE4 is associated with an increase in amyloid beta levels, neuroinflammation, and impaired neuronal function that appears to be a consequence of disrupted neuronal maintenance and repair (Liu et al., 2013). There are substantial reported sex differences in male and female APOE4 carriers. Female APOE4 carriers have almost 2x the risk of developing AD and were found to express greater brain hypometabolism and cortical thinning compared to female non-carriers, while the difference in male APOE4 carriers to non-carriers was much less (Altmann et al., 2014). APOE4 carrying females have greater risk than males between the ages 65–75 to develop AD and they are more likely to develop mild cognitive impairment between 55–70 (Neu et al., 2017). TBI also is associated with poorer outcomes in adult women over the age of 55 with the APOE4 allele, and again this relationship was not as robust in APOE4 expressing males (Eramudugolla et al., 2014, Ponsford et al., 2011). These sex differences necessitate that sex as a biological variable be implemented in studies investigating the APOE4 genotype, especially as it pertains to TBI outcomes and specifically pre-clinical TBI models.

Recent evidence supports that the possession of the APOE4 gene and alcohol use and/or abuse exacerbates cognitive deficits and advances AD onset. For example, the presence of the APOE4 allele and a history of heavy drinking are associated with earlier onset of AD (Panza et al., 2009, Solfrizzi et al., 2009, Zilkens et al., 2014) and dementia (Mukamal et al., 2003). In addition, the Original and Offspring cohorts of the Framingham Study has shown a correlation between alcohol consumption, APOE4, and risk of AD (Downer et al., 2014). Further, light and moderate alcohol consumption was associated with greater learning and memory decline among APOE4 carriers (Anttila et al., 2004, Downer et al., 2014, Kivipelto et al., 2008). Although this may be dependent on age, as there was no reported effect of midlife alcohol consumption in APOE4 carriers on learning and memory, but late life light and moderate drinkers exhibited a significant decline in learning and memory (Downer et al., 2014). Abstinence would be more beneficial among APOE4 carriers, as those who never use were at a lower risk of developing AD than those who drank 1 or more times a month, and increasing alcohol consumption is associated with increased risk of developing AD (Berkowitz et al., 2018, Anttila et al., 2004, Kivipelto et al., 2008). A more recent study involving a large population of nearly 400,000 participants showed a significant positive interaction between sex, high alcohol intake, and APOE4 on reasoning scores with male drinkers performing better than females (Lyall et al., 2019), however post-hoc analyses were not significant.

Despite the existence of numerous rodent TBI models, the effect of TBI on alcohol seeking behavior in APOE targeted replacement (TR) mice has not been studied and it is currently unknown whether APOE TR mice respond differently to intermittent or extended alcohol exposure. To address this, we aimed to establish a model that mimics a scenario of “drinking-abstinence-injury-drinking” and to utilize the IntelliCage system, a unique and innovative animal housing system that provides the ability to longitudinally monitor and study individual animal spontaneous activity in a home-cage environment, to test the hypothesis that APOE genotype influences voluntary alcohol consumption in the absence and presence of rmTBI in both male and female mice. Initially, we determined whether APOE genotype influences spontaneous activity and voluntary alcohol preference. Of note, female alcohol self-administration has consistently been reported to be higher both in home-cage environment and in operant response systems (Bertholomey et al., 2016, Bertholomey and Torregrossa, 2019, Priddy et al., 2017). Because drinking behavior varies with sex in both clinical and preclinical studies, it is important to examine the effect of TBI on alcohol use in both male and female APOE variant mice. This was followed by the assessment of APOE genotype influence on these measures after periods of abstinence and re-instatement after sham or rmTBI. The data presented indicate that APOE genotype differentially influences alcohol preference on the basis of sex. These results fill a gap in our knowledge regarding the effects of APOE genotype in combination with rmTBI on alcohol use disorder (AUD). Further understanding how APOE genotype influences alcohol use following trauma will be an important aspect in rehabilitative strategies to reduce the onset of cognitive and mental disorders associated with TBI.

Methods

Animals:

Male (N=24) and female (N=32) human APOE3 and APOE4 targeted replacement (TR) mice were used in this study. The TR mice were originally generated by gene targeting and backcrossed to C57BL6/J mice eight times as described previously (Sullivan et al., 1997). Homozygous APOE3 and APOE4 mice obtained from P. Sullivan were bred to generate the experimental mice, and maintained under a 12:12 light-dark cycle (lights on from 06:00h-18:00h) at the Durham VA Medical Center. Male mice were group housed from the time of weaning using Block Party cages (Ares Scientific, New York, NY) in order to avoid fighting when transferred to large group housing within the IntelliCage units. Male and female APOE TR mice (8–9 weeks old) were injected with radiofrequency identification (RFID) transponders up to 1 week prior to introduction to the IntelliCage and were randomly assigned to sham or rmTBI treatment groups. Mice had access to food and water ad libitum throughout the study. All experimental procedures were approved by the Durham VAMC Institutional Animal Care and Use Committee.

IntelliCage system:

The IntelliCage system (TSE Systems, Chesterfield, MO) used in this study consists of 2 computer controlled units (20.5 cm high and 55 × 38.5 cm at the base) that house up to 16 mice/unit; each unit is equipped with four conditioning corners containing a ring antenna that detects individual RFID implanted mice as they enter with an infrared sensor to detect presence. In addition, there are two computer-controlled doors which regulate access to 2 separate drinking bottles in each corner, 3 LED lights above each door for conditional stimulation, and valves to deliver brief air puffs for negative reinforcement. The experiments were implemented using Designer, Controller, and Analyzer software provided with the system. Three primary events were continuously recorded throughout the duration of the study: visits, nosepokes and licks. All data analysis was conducted as a function of these collected measurements using a variety of programmed modules for each experiment outlined in Figure 1. The free adaptation (FA) module provided access to all drinking bottles for an unlimited duration while the mouse remained in the corner. The nosepoke adaptation (NPA) module required a nosepoke to open the door and access to the bottle was available for 7 seconds, after which the door closed and the mouse was required to exit the corner before either of the drinking bottles could be accessed again. The NPA module was applied during intermittent alcohol access (IAA), where mice had 24h two-bottle choice between water and increasing concentrations of ethanol (EtOH) every other day (4–12% in 2% increments), and during extended alcohol access periods (EAA1 and EAA2) where mice had unlimited two-bottle choice between water and 12% EtOH for 6 weeks. During all alcohol access periods, the location of the alcohol bottle was signaled by a blue LED above the door upon entering the corner. In order to model binge-like drinking paradigms, an alcohol deprivation effect (ADE) and a drinking in the dark (DID) approach was used; for ADE mice were given repeated intermittent access to 12% EtOH for 3 days on/4 days off over a 4 week period and for DID mice were given 3h access to 12% EtOH beginning at 19:00h and 02:00h for 4 days. Water was available ad libitum. Motivation for 12% EtOH was determined using a progressive-ratio test where mice had to increase the number of nosepokes (2, 4, 6, 8, etc.) during a single visit in order to obtain access to the drinking bottle. The number of nosepokes was increased when a mouse performed 10 sets of each fixed ratio (FR) for motivation for water or 4 sets of each FR for alcohol. During the motivation for alcohol task, the FR to obtain water was set to 1. Motivation for EtOH was also assessed in males by examining the resistance to punishment. During this task, 2s after nosepoking at the side where %12 EtOH was located, mice received a brief air puff (0.5 s, 5 psi) delivered from an overhead port within the corner.

Figure 1.

Figure 1.

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Experimental timeline. Mice were introduced to the IntelliCages at 10 weeks of age and underwent a series of alcohol exposure paradigms (green), alcohol-free periods (white or gaps), and behavioral testing (yellow). Abbreviations: FA/NPA - free adaptation/nosepoke adaptation; EAA1 and EAA2 - extended alcohol access; rmTBI - repeated mild traumatic brain injury; ADE - alcohol deprivation effect; PR - progressive ratio; DID - drinking in the dark; LDB/EPM - light-dark box/elevated plus maze; SRL - serial reversal learning.

Traumatic brain injury induction:

Three weeks following removal of 12% EtOH (at approximately 4 months of age), mice received sham or closed-head rmTBI using a custom designed pneumatic impactor device (S&D Tool, Durham, NC). Mice were removed from the IntelliCage units and transferred to the TBI procedure room using large transport cages. Mice were anesthetized in 4.5% isoflurane for two minutes in a sealed induction chamber and then quickly placed on a foam block with cutouts that stabilize the lateral movement of the head. Mice were positioned directly below the impact tip, which was aligned along the mid-line (left to right) and mid-way between the back of the orbits and the back of the intraparietal bone. The pneumatic impact cylinder (Parker Hannfin, Ltd., Cleveland, OH, part # 1.06 CKDX SR B 1.00) was fitted with a 50 duro, 9 mm silicone sphere at the end of the impactor rod tip which distributes energy across the scalp and skull and prevents skull fracture. The impactor was activated using pressure delivered from a compressed nitrogen gas cylinder connected to a solenoid that was opened using a 100 ms, 24V stimulation pulse and imparted a strike velocity of 3.5–4.0 m/s and a 4 mm displacement beyond the surface of the head. Immediately following impact, the mouse was removed from the foam block and placed supine in a custom designed holder and the latency to recovery of the righting reflex (RoRR) was recorded. Upon returning to the prone position, the mouse was placed in a new transport cage to allow for further recovery. After all mice had received treatment, they were returned to the IntelliCage units and activity was monitored using the FA module. The procedure was repeated at 24h intervals for three consecutive days. For sham treated animals, the same procedure was applied, except that the animal was not placed directly under the impactor while it was discharged. This procedure is similar to the CHIMERA method (McNamara et al., 2020) and the resulting injury is considered mild based on the absence of skull fracture, hemorrhage or other gross anatomical lesions (no tissue loss), as well as a brief loss of consciousness and mild motor impairments. Mortality resulting from the rmTBI procedure was < 5% (1 female out of 28 rmTBI treated mice).

Behavioral Assessment:

Anxiety-like behavior was measured 2 months following the rmTBI procedure and at the end of the 6 week alcohol re-introduction phase (EAA2). Light/Dark box. Mice were placed in the light portion of the light/dark box apparatus equipped with an overhead camera attached to a computer and allowed to explore the area for 5 minutes. The total time spent in the light and dark compartments and the distance travelled in the light field were analyzed. Elevated Plus Maze. One day (for females) or three days (for males) following the light/dark box task, mice were placed in the center of the apparatus equipped with an overhead camera attached to a computer and allowed to explore the area for 5 minutes. The total time and distance travelled in the open and closed arms were analyzed. Behavior was recorded and analyzed using AnyMaze v. 3.1 tracking software (Stoelting Co., Wood Dale, IL). Serial Reversal Learning. Cognitive flexibility was assessed by assigning a single correct corner to each mouse and alternating to the opposite corner every 24h. The % Error of visits with nosepokes was analyzed in 3h bins between 18:00–06:00 to assess learning over the course of each day for a total of 6 days. Further, the % Error during the first 3h bin on each reversal day were compared to determine a rule learning effect.

Data Analysis and Statistics:

IntelliCage data was processed using Analzyer software and exported for analysis using Excel (Microsoft Corporation, Albuquerque, NM) and GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). Primary measurements within the IntelliCage included the number of visits, nosepokes and licks, serving as the basis of subsequent analyses. Unless otherwise noted, the data was exported in 12h bins that corresponded to the light and dark phases of the housing light cycle and the reported values represent the combined 24h period from 18:00–18:00 (dark phase plus light phase). Alcohol preference was calculated by dividing the number of EtOH licks by the total number (water plus EtOH) during all exposure periods. The dose of EtOH consumed was calculated by estimating a volume of 3 μL per lick. For motivation, data is presented as progressive ratio breakpoint reached or the % of punished nosepokes for access to EtOH. Data is presented as mean±SEM. Statistical analysis was performed using a two-way ANOVA (genotype X TBI) or a 3-way repeated measures ANOVA (genotype X TBI X time as the repeated factor). Significant ANOVA results were followed by Tukey’s HSD to determine specific group mean differences (p<0.05).

Results

Voluntary drinking behavior in APOE TR mice

Male (cohort 1) and female (cohort 2) human APOE TR mice were separately and consecutively provided long-term housing within IntelliCage units and subjected to a number of experimental conditions according to the schedule outlined in Figure 1. Prior to introduction to the IntelliCage, mice were randomly assigned to one of the following treatment groups: E3 sham, E3 TBI, E4 sham, and E4 TBI (N=6 per group for males and N=8 per group for females). Upon initial introduction to the IntelliCage units, mice were subjected to a FA and NPA module for 3 days and 1 day, respectively, in order to track spontaneous activity prior to any experimental manipulation. There were no genotype differences in the number of total visits, nosepokes, licks, % visits with nosepokes, % visits with licks, and % nosepokes with licks; however, there was a significant main effect of sex for all of these measures with females displaying significantly greater visits and nosepokes but fewer licks, % visits with licks, % visits with nosepokes, and % nosepokes with licks than males (Supplemental Figure S1).

One day following NPA mice were introduced to EtOH using a two-bottle voluntary choice between water (no light cue above door) or increasing % of EtOH between 4–12% at 2% increments every other day (blue light cue above door) within each of the four operant corners. When 12% EtOH level was reached, mice had unlimited extended alcohol access (EAA1) with a choice between water and 12% EtOH for 6 weeks to develop a long-term dependence. The voluntary preference for EtOH per day during the introduction period and the weekly average % EtOH preference for 12% EtOH during EAA1 are presented in Figure 2A and 2B, respectively. During the introduction period there was a significant concentration X sex interaction (F(4,208)=17.96, p<0.001) as well as a significant concentration X genotype interaction (3-way RM-ANOVA, F(4,208)=2.77, p<0.0282). During EAA1 a 3-way RM-ANOVA revealed a significant sex X genotype interaction (F(1,52)=17.90, p<0.0001), a significant week x sex interaction (F(5,260)=6.061, p<0.0001), and a significant week X genotype interaction (F(5,260)=3.704, p=0.003). Post-hoc analysis revealed a significant decrease in alcohol preference among APOE3 males in weeks 2–6 compared to week 1 (Tukey’s post-hoc, p < 0.05). There was also a significant genotype difference in males during weeks 3–6. To compare groups without the repeated measures factor, individual EtOH preferences and doses were averaged across the 6 week access period and are presented in Figure 2D–E, again revealing a significant sex X genotype interaction for both preference (F(1,52)=17.90, p<0.0001) and dose (F(1,52)=10.5, p=0.0021). Post-hoc analysis resulted in significant differences between all groups except for the dose between female APOE3 and APOE4 mice (Tukey’s HSD, p<0.001). Overall, females show a significantly higher preference and consume greater amounts of alcohol than males while APOE genotype differentially influences preference depending on sex where APOE4>APOE3 in males and APOE3>APOE4 in females.

Figure 2.

Figure 2.

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Voluntary alcohol consumption in adult male and female APOE TR mice. (A) Average daily preference during initial introduction to EtOH where mice had 24h two-bottle choice access to increasing concentrations from 4–12% in 2% increments every other day. (B) Average weekly % EtOH preference during extended alcohol access (EAA1) where mice had unlimited two-bottle choice access to 12% EtOH or water for a duration of six weeks. Preference significantly decreased in male APOE3 mice during weeks 3–6 relative to week 1 and to all other groups (*p<0.05). Individual values of % EtOH preference (C) and corresponding dose (D) averaged across the 6 week EAA1 period resulted in significant sex X genotype interactions (p<0.0001, post-hoc *p<0.05, ****p<0.0001.). Data are presented as mean ± SEM (n=12/group for males and n=16/group for females).

Abstinence and TBI spontaneous activity.

Following the EAA1 period, alcohol was removed for 3 weeks, and mice were maintained using the FA module. The spontaneous activity averaged during the first week of the EtOH-free period is presented in Supplemental Figure S1 and this activity remained stable for the 3 week duration. Compared to pre-EtOH access, there was a significant decrease in visits and nosepokes and an increase in licks, % visits with licks and % nosepokes with licks for all groups. Mice then underwent sham or rmTBI for 3 consecutive days at 24h intervals. The recovery of righting reflex (RoRR) in response to sham or injury, and the average number of corner visits and licks per day post-injury relative to baseline activity before injury are presented in Figure 3. There was a genotype-independent significant main effect of TBI on RoRR in males (F(1,20)=23.09, p<0.0001) and a TBI X day interaction in females (F(2,54)=6.229, p=0.0037), with RoRR significantly greater after the second impact compared to the first (Tukey’s post-hoc, p<0.05). Subsequent analysis comparing the effects of in rmTBI only subjects revealed that females have significantly greater RoRR compared to males (3-way ANOVA, F(1, 23)=29.03, p<0.0001). Measures of spontaneous activity relative to baseline presented in Figure 3C–F revealed a significant main effect of day on the number of corner visits during the rmTBI procedure (3-way repeated measures ANOVA, p<0.0001) for both sexes. While there was no effect of rmTBI on corner visits in males, there was a significant effect of rmTBI in females (F(1,27)=12.82, p=0.0013), with injured mice exhibiting a significant decrease in activity. Corner visits in both males and females returned to baseline levels following the rmTBI procedure, but there was a subsequent drop in males following recovery. In addition, there was no effect of rmTBI on the number of bottle licks in males, while there was a significant genotype-independent effect of rmTBI in females (F(1,27)=11.67, p=0.002).

Figure 3.

Figure 3.

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Recovery of righting reflex (RoRR) and spontaneous activity post-rmTBI. Mice received sham or mild TBI for 3 consecutive days at 24h intervals. rmTBI significantly increased RoRR compared to sham in (A) males and (B) females (#main effect of TBI, p<0.0001). In females RoRR increased on Day 2 compared to Day 1 (*p<0.05). Average daily corner visits (C-D) and average daily bottle licks (E-F) within the IntelliCage presented as the percentage relative to baseline (B), during rmTBI (T), and during recovery (R). In both sexes, the rmTBI and sham procedures significantly reduced the number of corner visits relative to baseline (# main effect of procedure, p<0.05), but there was a significant effect of rmTBI compared to sham treated mice in females on all days with TBI and the first day of recovery (*p<0.05). rmTBI also significantly impacted the number of licks relative to baseline in female (#main effect of procedure, p<0.0001;*p<0.05 relative to sham), but not male mice. Data are presented as mean ± SEM (n=6–8/group).

Post-TBI reintroduction to alcohol

Five days following the last TBI, 12% EtOH was re-introduced for unlimited two-bottle choice EAA2 for 6 weeks. The average weekly % EtOH preference in males and females are presented in Figure 4A–B, respectively. In males, 3-way RM-ANOVA revealed a main effect of time (F(2.5,50)=28.76, p<0.0001) with a gradual decrease in preference over the access period. In females there was also a significant main effect of time (F(2.7,74)=16.34, p<0.0001), but the decrease in preference did not occur until week 5 of access. There was no significant effect of rmTBI on alcohol preference as a function of genotype in either sex. In order to directly compare males and females, the % EtOH licks and doses were averaged across the entire 6-week period (Figure 4C–D). A 3-way ANOVA revealed significant main effects of sex for preference (F(1,47)=43.96, p<0.0001) and dose (F(1,47)=22.97, p<0.0001) with females being greater than males. Additional 3-way RM-ANOVA was conducted within each sex to compare the EAA1 and EAA2 dosages and revealed TBI-independent main effects of time in females (F(1,27)=112.8, p<0.0001) and a time X genotype interaction for males (F(1,20)=12.54, p=0.002) (Supplemental Figure S2). Females from all groups consumed significantly less EtOH during EAA2, while only male APOE4 rmTBI mice had a significant decrease (Tukey’s post-hoc, p<0.05).

Figure 4.

Figure 4.

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Re-introduction to 12% EtOH (EAA2) beginning 5 days post-rmTBI. There was a significant main effect of time for the average weekly 12% EtOH preference in males (A) and females (B) (p<0.0001). Averages of 12% EtOH preference (C) and dose (D) across the 6 week EAA2 period resulted in a significant main effect of sex (#p<0.0001), with females continuing to display a higher % EtOH preference and dose than males independent of genotype or rmTBI. Data are presented as mean ± SEM (n=6–8/group).

Alcohol deprivation effect (ADE), drinking in the dark (DID), and motivation

Following the six-week EAA2 re-introduction period, EtOH was removed for an additional 4 weeks and mice were subsequently given repeated intermittent access to 12% EtOH for 3 days on/4 days off over a 4 week period in order to determine if this binge-like access would have a greater influence on EtOH preference. A 3-way RM-ANOVA of the data presented in Figure 5A resulted in a significant week x genotype interaction (F(3,60)=2.832, p=0.0458) for males, where APOE4 mice consumed greater EtOH. In addition to a significant week x genotype interaction (F(3,78)=3.706, p=0.015) in females shown in Figure 5B, there was also a signinificant genotype x TBI interaction (F(1,26)=12.12, p=0.0012). A 3-way ANOVA of %EtOH preference averaged across the 4 weeks of EtOH access periods presented in Figure 5C revealed a significant genotype X sex interaction (F(1,46)=16.72, p=0.0002) as well as a significant genotype X TBI interaction (F(1,46)=4.84, p=0.0329). A 3-way ANOVA of dose averaged across the 4 weeks of EtOH access periods presented in Figure 5D revealed a genotype X sex X TBI interaction (F(1,46)=5.803, p=0.02). Female APOE3 sham mice had greater preference and dose compared to sham and rmTBI-treated male APOE3 mice and female sham APOE4 mice.

Figure 5.

Figure 5.

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Alcohol preference during binge-drinking paradigms. An alcohol deprivation effect (ADE) was used to measure the average 12% EtOH preference when mice were given 3 consecutive days of access followed by 4 days of abstinence for 4 sessions. Preference for EtOH remained stable across time in males (A) while there was a significant main effect of weekly session in females (B) with preference increasing upon subsequent exposure compared to the first session. There were significant genotype X sex and genotype X TBI interactions for the average 12% EtOH preference (C) and dose (D) across all 4 access sessions. (E-F) A drinking in the dark (DID) paradigm was used to measure the average 12% EtOH preference when mice were given limited access for two, 3h sessions/night (19:00–22:00 and 02:00–0:500) for 4 days (total 8 access sessions). Preference for alcohol steadily increased upon subsequent exposure sessions for APOE4 males (E) while it remained stable in females (F). (G-H) There were significant genotype X sex and genotype X TBI interactions for the average % EtOH preference and dose across all 8 sessions of access. Data are presented as mean ± SEM (n=6–8/group). Female APOE3 sham mice had greater preference and dose compared to male APOE3 mice and female sham APOE4 mice (*p<0.05).

Approximately 1–3 months following the ADE sessions, mice were given limited access to 12% EtOH during two 3h sessions into the night phase (19:00–22:00 hrs and 01:00 to 04:00 hrs) over four days for a total of 8 access sessions in order to determine EtOH preference using a limited access DID paradigm. A 3-way RM-ANOVA of the data presented in Figure 5E resulted in a significant main effect of genotype (F(1,20)=9.812, p=0.0052) for males, while in Figure 5F there was a significant genotype X TBI interaction for females (F(1,216)=85.25, p<0.0001). A 3-way ANOVA of %EtOH licks averaged across all EtOH access sessions in Figure 5G revealed a significant genotype X sex interaction (F(1,46)18.04, p=0.0001) as well as a significant genotype X TBI interaction (F(1,46)=8.514, p=0.0054). A 3-way ANOVA of dose averaged across all EtOH access sessions shown Figure 5H revealed a significant genotype X sex X TBI interaction (F(1,46)=5.685, p=0.0213). Again, female APOE3 sham mice had greater preference and dose compared to sham and rmTBI-treated male APOE3 mice and female sham APOE4 mice.

Motivation for EtOH was assessed using a progressive ratio (PR) test where increasing nosepokes performed within a single visit and timeframe (5 seconds) were required to gain access to water (Figure 6A) or 12% EtOH (Figure 6B) over the course of one day. The PR task for water was performed on the first day to serve as a control to demonstrate that mice were able to perform the task, followed by the availability of EtOH on the second day. For motivation for water there was a significant sex X genotype X TBI interaction (F(1,46)=6.211, p=0.0164) for the maximum FR reached, with males reaching a higher ratio than females. For motivation for EtOH, there was a significant sex X genotype interaction (F(1,46)=20.34, p<0.0001) and a significant genotype X TBI interaction for the maximum FR reached for 12% EtOH (F(1,46)=4.569, p=0.0379). Post-hoc analysis revealed significantly higher motivation for male APOE4 rmTBI mice compared to all other groups (Tukey’s post-hoc, p<0.05) except for male APOE4 shams. Resistance to punishment was examined in male mice where a brief air puff was delivered 2 seconds after a nosepoke at the side where the 12% EtOH was located. A 3-way RM-AONVA of the data presented in Figure 6C resulted in a significant genotype X TBI interaction (F(1,20)=17.93, p=0.0004) and a significant day X genotype interaction (F(4,80)=5.21, p=0.0009). Although APOE4 rmTBI mice performed more nosepokes at the punished side than the other groups, these differences were not significant on any specific day after post-hoc correction (p>0.05).

Figure 6.

Figure 6.

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Male APOE4 mice exhibit motivation for EtOH. (A) Mice were first subjected to a progressive ratio task in which motivation for water was assessed. There was a significant main effect of sex (#p<0.0001) on the PR breakpoint reached, with males reaching a higher PR than females. (B) For motivation for 12% EtOH there was a significant sex X genotype interaction (#p<0.0001) on the PR breakpoint reached with post-hoc analysis revealing APOE4 males exhibit significantly higher motivation than all other groups independently of rmTBI (*p<0.005). Resistance to punishment was examined in male mice (C) and resulted in a significant genotype X TBI interaction (p<0.005), with post-hoc anlysis revealing APOE4 mice performing more nosepokes than E3 mice at the punished side where 12% EtOH was located. Data are presented as mean ± SEM (n=6–8/group).

rmTBI influences risk-taking behavior but not spatial cognition

At the end of the extended reintroduction period, while access to 12% EtOH was still available, mice were briefly removed from the IntelliCage and tested using the light/dark box and the elevated plus maze to assess anxiety-like behavior. The data presented in Figure 7A–B revealed a main effect of TBI for the time spent and the distance traveled in the light zone with rmTBI-treated mice spending more time (F(1,46)=15.37, p=0.0003) and traveling greater distances (F(1,46)=25.68, p < 0.0001) compared to sham-treated mice. Similarly, rmTBI-treated mice spend more time (F(1,46)=39.82, p < .0001) and travel greater distances (F(1,46)=40.7, p < .0001) in the open arms of the elevated plus maze (Figure 7C–D). There was no significant effect of sex or genotype within any of these measures.

Figure 7.

Figure 7.

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rmTBI reduces anxiety-like traits independent of sex and APOE genotype. Anxiety-like behavior was assessed at the end of the EAA2 period. A 3-way ANOVA revealed a main effect of rmTBI (#p<0.005) for the time spent (A) and distance traveled (B) in the light compartment of the light/dark box task and the time spent (C) and distance traveled (D) in the open arms of the elevated plus maze. Data are presented as mean ± SEM (n=6–8/group).

Following the DID procedure, during a period when EtOH access was not available, a serial reversal task was performed within the IntelliCage to assess spatial memory and cognitive flexibility. At the start of the task, mice were assigned to a single corner in order to gain access to water, and the % Error of visits with nosepokes was measured within 3h bins during the 12h dark phase. The following day, before the start of the dark phase, the correct corner was switched to the opposite side and this reversal continued for a total of 6 days. A 3-way RM-ANOVA of the data presented in Figure 8A–B resulted in a significant main effect of time in the reduction of % Error of visits with nosepokes over the course of four 3h time bins during the 12h dark phase each day for all groups in each sex (p<0.0001), thus demonstrating that mice were able to learn the place of the rewarded corner after each daily reversal. Since there were no differences among any of the groups within each sex, data were pooled in order to compare sex differences for the % Error during the first 3h bin of each daily reversal, which reflects a rule learning effect, and is presented in Figure 8C. A 2-way RM-ANOVA resulted in significant main effects of time (F(5,318)=22.04, p< 0.0001) and sex (F(1,318)=50.94, p<0.0001), with post-hoc analysis revealing males performing better than females on reversals 3–6 (p<0.05).

Figure 8.

Figure 8.

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Male APOE mice display greater cognitive flexibility than females independently of genotype and rmTBI. Cognitive flexibility was assessed using the serial reversal place learning task within the IntelliCage in the absence of EtOH access. Mice were assigned to a single correct corner in order to gain access to water and the % Error of visits with nosepokes were calculated and plotted across the 12h dark phase of each day in four 3h time bins. Each subsequent day mice were assigned to the opposite corner and this reversal was repeated 5x for a total of 6 days. The % Error significantly decreased across the four time bins for all groups in both males (A) and females (B) over the course of each 12h dark phase for all 6 days independently of genotype or rmTBI (3-way RM-ANOVA, p<0.0001). (C) A rule learning effect is illustrated by plotting the average % Error (collapsed across genotype and rmTBI) during the first 3h time bin of each day among males and females. A 2-way RM-ANOVA revealed significant main effects of day and sex (p<0.0001) with females having significantly greater errors than males on days 3–6 (*p<0.05). Data are presented as mean ± SEM (n=6–8/group).

Discussion

This is the first preclinical study performed to characterize voluntary alcohol consumption as a function of APOE genotype using the APOE TR mouse model. To address this, we aimed to establish a model that mimics a scenario of “drinking-abstinence-injury-drinking” to determine if the APOE genotype influenced alcohol consumption throughout this trajectory. Initially, we measured alcohol preference as a function of APOE genotype and sex during an intermittent access period to acclimate the mice to increasing EtOH concentrations and then during an extended drinking period at a high EtOH concentration to establish dependence. Three weeks after an alcohol-free period mice received sham or rmTBI and were then subsequently provided access to 12% EtOH under a variety of experimental paradigms over the course of several months.

Results from this study revealed several major findings. First, our data showed a significant sex X genotype interaction for alcohol preference during both the initial extended access period (EAA1) and during the re-introduction period (EAA2). Contrary to our hyspothesis, there was no effect of rmTBI during the EAA2 period in either sex or genotype. However, during the ADE and DID binge-like drinking paradigms, a genotype X rmTBI interaction was observed in females. Second, rmTBI resulted in a genotype-independent anxiolytic behavioral phenotype but did not affect spatial learning. However, since males outperformed females in this task, it is possible that the higher EtOH consumption in females throughout the duration of the study may have contributed to this difference. Finally, there is a significant sex by genotype interaction for motivation for EtOH where APOE4 males show the greatest motivation in the progressive ratio task, and a futher effect of rmTBI in resistance to punishment. Surprisingly, while female mice have a significantly greater preference for EtOH than males under free-choice access, they exhibit very little motivation for it, with only a few subjects responding to a small incremental increase beyond two nosepokes required for access. This suggests that females may be more likely to abstain when alcohol is not easily accessible.

During reintroduction to EtOH after the abstinence period and following the sham or rmTBI procedure (EAA2), there was a significant reduction in drinking compared to pre-injury (EAA1) in all treatment groups except male APOE3 mice, which displayed a low preference to begin with. Furthermore, preference appears to diminish over time during both extended access periods, particularly in males. In agreement with this, it has been reported that continuous alcohol access paradigms consistently show lower intake than intermittent access paradigms (Crabbe et al., 2011, Griffin, 2014, O’Dell et al., 2004, Spanagel, 2000). Another factor that could play a role in the decreasing preference seen during EAA2 and subsequent access is the age of the mice. Differences in several measures such as spatial learning and memory (Bour et al., 2008), neuronal structure and function (Klein et al., 2014, Klein et al., 2010), and metabolic and inflammatory processes (Dumanis et al., 2013, Zhu et al., 2012) have been noted with aging and also as a function of genotype in the APOE TR mice, and it is possible that these processes are contributing to the differences and/or changes in alcohol preference over the timecourse seen in this study.

During the ADE paradigm, which induces binge-link drinking patterns, male APOE4 mice showed a TBI-independent significantly higher preference than male APOE3 mice over time and this preference did not diminish with multiple repeat exposures. This indicates that APOE4 male mice continue to consistently consume alcohol across the lifespan whereas male APOE3 mice diminish intake over time. In contrast, female APOE3 sham mice displayed greater preference for EtOH than APOE3 rmTBI mice while preference in APOE4 females showed an opposite trend with an increase in rmTBI mice compared to shams. These results suggest that the interaction of TBI and genotype influence alcohol preference differently in male and female APOE TR mice. While continued unlimited access is used to develop an EtOH-dependent phenotype, ADE has also been shown to replicate relapse-like drinking due to repeated deprivation phases and lead to compulsive drinking patterns (Vengeliene et al., 2014). Thus, it appears that in order to truly capture alcohol seeking behavior in the APOE TR mouse model, the ADE and/or DID model should be used in order to increase or sustain preference, particularly in males.

Until recently the majority of studies measuring alcohol preference have been performed in singly housed animals. This social isolation likely adds a stressful component that may influence EtOH consumption (Lopez and Laber, 2015). Another study showed that single-housed mice consumed more EtOH than group-housed mice (Holgate et al., 2017). The IntelliCage system is a unique and innovative animal housing system that provides the ability to monitor and study individual animal spontaneous activity via implanted radiofrequency chips, as well as learning and memory over extended time periods in a social environment (Dere et al., 2018, Kiryk et al., 2020). Further, it enables the simultaneous testing of up to 16 mice per unit, with mice residing in a home-cage and a social environment with no experimenter handling, reducing the confounding effects of experimenter stress and social isolation typically seen in animal models of alcohol consumption. In general, our data is in agreement with other published studies assessing voluntary alcohol drinking in the IntelliCage where mice readily consumed both low and high concentrations of alcohol, exhibited sex differences in EtOH preference with females drinking more, and displayed a range of preference between subjects (Koskela et al., 2018, Radwanska and Kaczmarek, 2012, Smutek et al., 2014), thus lending further support for the reproducibility of alcohol seeking behavior using the IntelliCage. Our data is also in agreement with studies assessing the effects of TBI on EtOH preference in male C57BL/6 mice which have shown repeated blast TBI lowered the mean EtOH intake (of 20% EtOH) over a 24h period (Schindler et al., 2021) and that moderate impact TBI lowered intake (of 15% EtOH) using a DID paradigm (Lowing et al., 2014).

Results from behavioral assessments performed in the IntelliCage tend to track closely with those generated from traditional behavioral assessments including circadian, spatial cognition and anxiety-like behavior. For example, anxiety levels in mice treated with an anxiolytic drug found that a modified Vogel conflict paradigm in the IntelliCage showed a decrease in anxiety-like behavior which was confirmed by results from the more traditional elevated plus maze (Kiryk et al., 2020). This enriched environment, however, could likely contribute to the lack of spatial deficits resulting from rmTBI observed in this study. Previous studies have reported improved spatial memory performance in experimentally manipulated or aged mice (Huo et al., 2012, Konopka et al., 2010, Mechan et al., 2009). In addition, learning deficits may be attenuated when experimental groups are co-housed (Lipina and Roder, 2013), as was the case in this study where rmTBI mice were cohoused with sham injured mice. For example, human amyloid precursor protein transgenic mice were able to learn the correct corner for receiving access to water (place learning task) when housed with wild type mice, but displayed impairments when housed separately (Kiryk et al., 2011). Several studies have also shown a neuroprotective effect of EtOH on cognition. For example, the influence of EtOH on recovery from rmTBI in mice using a weight-drop model and EtOH access for 4 weeks suggested that EtOH might have a protective effect on recovery if consumed before, but not after the trauma (Baratz et al. 2010). A clinical study also showed that APOE4 was not associated with cognitive deficits during EtOH withdrawal (Wilhelm et al., 2005). Another study showed that light drinking in male APOE4 carriers had a protective effect on cognitive function (Carmelli et al., 1999). In rats, behavioral flexibility remained intact in high EtOH-preferring rats (McMurray et al., 2014). Finally, it is important to note that the spatial learning task was conducted in the absence of EtOH and therefore it is possible that performing this task during the presence of EtOH could lead to deficits in cognitive flexibility or other forms of cognitive learning. Future studies will be aimed at assessing cognitive learning in the presence of EtOH as well as under conditions where sham and TBI mice are housed separately.

In contrast to the spatial learning task within the IntelliCage, we found a significant main effect of rmTBI in tasks that were conducted outside of the socially housed environment. rmTBI mice spent more time and travelled greater distance in the light compartment of the light/dark box and within the open arms of the elevated plus maze. This finding is consistent with a risk-taking behavioral phenotype, which has also been observed in previous studies (Mouzon et al., 2014, Mannix et al., 2014). Despite this increased risk taking, it does not appear to predict or correlate with an increase in alcohol intake.

The limitations of this study include the small sample size when broken down into sham and TBI as well as a moderate variability in EtOH preference within these groups. Nevertheless, the differences were large enough to result in several statistically significant findings, especially with regard to sex differences. Future studies should be sufficiently powered to potentially categorize mice into high and low drinkers. In this study, we did not measure BAC levels and are therefore unable to determine the precise degree of intoxication. Based on a previous study of EtOH self-administration in mice where BAC levels were measured and correlated with EtOH dosage, it was estimated that there is an approximate 15 mg/dL rise in BAC for every 1 g/kg of EtOH consumed in a 3.5h time period (Blegen et al., 2018). Extrapolating from this, we estimate that BAC levels ranged between 0–60 mg/dL under the 3h access DID paradigm. Finally, in humans, EtOH consumption following TBI is generally low within the first year of injury (Pagulayan et al., 2016). Therefore, it is possible that re-introduction only 5 days following the last TBI (EEA2) was too soon and that the mice quickly developed an aversion to the effects of EtOH during this acute period, particularly in males.

Future studies aimed at determining the effects of EtOH exposure during adolescence will also be critical in furthering our understanding of how mTBI influences EtOH consumption and overall brain function later in life on the basis of APOE genotype. Adolescence is a critical period in shaping the brain or priming the brain for addictive behaviors in adulthood and EtOH consumption in adolescence can significantly influence brain development, cognition, personality and executive function. Our previous work in adolescent rats demonstrated that EtOH exposure during adolescence leads to long-lasting structural and functional alterations in the hippocampus (Fleming et al., 2013, Risher et al., 2015). A recent study in mice using the DID model showed that adolescent binge drinking modulates ethanol-related anxiety behavioral effects later in life as well as enhances drinking in adulthood (Younis et al., 2019). Alcohol drinking in adolescence also increases consumption in adulthood in rats (Amodeo et al., 2017). However, the opposite was shown in a recent paper in rats exposed to ethanol vapor during adolescence (Nentwig et al., 2019). This suggests that the method of exposure may be a factor for addictive behaviors in adulthood. A study by Weil et al. found that female mice injured as juveniles, but not adults, displayed greater EtOH self-administration in adulthood (Weil et al., 2016a, Weil et al., 2016b). In this study we did not measure any brain changes such as receptor expression, inflammatory markers or synaptic adaptations that may underlie the differences associated with EtOH preference or long-term consumption. Since this study examined alcohol preference across the lifespan and under a variety of conditions, and that the level of intake varied significantly among groups, it would be difficult to attribute any measured differences to a specific factor. To better address this, future studies are warranted to determine how sex, APOE genotype, rmTBI and alcohol interact to influence brain changes at specific time points as well as under controlled EtOH dosages. Finally, additional studies are warranted to examine drinking behavior of APOE variant mice in single housing conditions to determine the effects of APOE genotype in this isolated context.

Conclusion

This is the first preclinical study assessing EtOH self-administration as a function of APOE genotype, sex and rmTBI. Alcohol use following injury can diminish recovery and rehabilitation and lead to a myriad of neurological disorders and psychiatric dysfunction. This EtOH-associated effect can be influenced by numerous factors such as a history of alcohol use prior to injury, social and environmental (psychosocial) factors, sex, and genetics. Based on the findings in this study, these variables should be taken into consideration when assessing the negative impact of EtOH consumption on mental health following mild TBI.

Supplementary Material

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fS1

Acknowledgements

This work was supported by a Department of Veterans Affairs (VA) Rehabilitation Research and Development Merit Review Award 5I01RX002335 (RCK), a VA Biomedical Laboratory Research and Development Merit Review Award 5I01BX001271 (SDM), and the VISN6 Mid-Atlantic Mental Illness Research, Education and Clinical Center (RCK, SDM). We would like to thank Kelsey R. Smith, PhD for her technical assistance in this study.

Funding Information: Department of Veterans Affairs, Rehabilitation Research and Development Merit Review Award 5I01RX002335 and Biomedical Laboratory Research and Development Merit Review Award 5I01BX001271; VISN6 Mid-Atlantic Mental Illness Research, Education and Clinical Center.

Footnotes

Disclaimer

The views expressed in this scientific manuscript are those of the authors and do not reflect the official policy or position of the U.S. government or the Department of Veterans Affairs.

Data Sharing

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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fS2
NIHMS1741432-supplement-fS2.tif (189.4KB, tif)
fS1
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