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. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: Alcohol Clin Exp Res. 2022 May 11;46(7):1282–1293. doi: 10.1111/acer.14853

Sex-related differences in pattern of ethanol drinking under the intermittent-access model and its impact on exploratory and anxiety-like behavior in Long-Evans rats

Breanne E Pirino 1, Cydney R Martin 1, Brody A Carpenter 1, Genevieve R Curtis 1, Christina M Curran-Alfaro 2, Shanna B Samels 1, Jacqueline M Barker 2, Anushree N Karkhanis 3, Jessica R Barson 1
PMCID: PMC9357056  NIHMSID: NIHMS1803488  PMID: 35491472

Abstract

Background:

While men in the United States consume more alcohol than women, rates of drinking are converging. Nevertheless, females remain underrepresented in preclinical alcohol research. Here, we examined rats for sex-related differences in patterns of ethanol drinking and effects of this drinking on exploratory and anxiety-like behavior.

Methods:

Adult male and female Long-Evans rats were given 20% ethanol under the intermittent-access two-bottle-choice paradigm. Their intake was measured daily for the first 7 weeks. During the 8th week, intake was measured over the 24 hours of daily access. During the 9th week, they, along with ethanol-naive controls, were tested prior to daily access in a novel chamber, light-dark box, and hole board apparatus. During the 10th week, blood ethanol concentration (BEC) was assessed after 30 – 40 minutes of access.

Results:

Compared to males, females overall demonstrated higher ethanol intake and preference across all access weeks, although only half of them drank significantly more than males. Across 24 hours of daily access, both sexes had their highest intake in the first 30 minutes and their lowest in the middle of the light phase of the light/dark cycle. Despite their greater ethanol intake, females did not show significantly different BECs than males. In behavioral tests, females compared to males showed less vertical time in a novel activity chamber, more movement between chambers in a light-dark box, and more nose pokes in a hole-board apparatus. While a history of ethanol drinking led to a trend for reduced vertical time in the activity chamber and increased chamber entries in the light-dark box, this was not sex-dependent.

Conclusions:

These results suggest that female and male rats could both be tested for acute effects of ethanol after 30 minutes of daily access, but that nuanced considerations should be made in designing these experiments and interpreting the findings.

Keywords: female, hole-board apparatus, light-dark box, novel chamber, male

While men in the United States consume more alcohol than women, the rates of drinking are converging between the genders (Hasin et al., 2019, Shmulewitz et al., 2021, White, 2020). Notably, despite their less frequent and less heavy drinking, women are more susceptible than men to a range of alcohol-related morbidities, including both physical diseases and affective disorders (Shmulewitz et al., 2021, White, 2020). This suggests that it is important to also examine females, and to compare them to males for various aspects and effects of ethanol drinking; however, regardless of the increasingly similar alcohol use, and seemingly worse related harms in women, female subjects have historically been, and continue to be, underrepresented in preclinical alcohol research (Guizzetti et al., 2016).

Notwithstanding the relative paucity of research on sex-related differences in relation to ethanol drinking, several preclinical studies have examined quantitative differences between males and females in levels of ethanol drinking. Under two-bottle-choice ad libitum access paradigms, females have been found to drink more ethanol than males over a range of ethanol concentrations and rodent strains, including Long-Evans rats, Wistar rats, selectively-bred alcohol-preferring AA and P rats, and WSC-1 mice (Bell et al., 2011, Eriksson, 1972, Priddy et al., 2017, Tambour et al., 2008). A few, but not all, studies have also found that females show elevated ethanol intake and preference under intermittent-access 16 – 20% ethanol two-bottle-choice paradigms (Li et al., 2019, Loi et al., 2014, Morales et al., 2015, Priddy et al., 2017, Quadir et al., 2022, Schramm-Sapyta et al., 2014, McNamara and Ito, 2021), with no effects of estrous cycle stage (Li et al., 2019, Priddy et al., 2017). To our knowledge, no studies have examined patterns of ethanol drinking under the intermittent-access model across the 24-hours of daily access to determine if there are qualitative sex-related differences in ethanol drinking. These patterns could reveal, for example, if there are differences in drinking early in the session (“front loading”) which could indicate disparities in motivation, or if there are differences in duration between large bouts, which could indicate disparities in reward value (Darevsky et al., 2019), among other factors. Interestingly, despite the higher ethanol intake of female rodents, female blood ethanol concentrations (BECs) are generally found not to be significantly different than those of males (Kulkosky, 1980, Lancaster and Spiegel, 1992, Li et al., 2019), suggesting that the behavioral impact of drinking might not be sex-dependent.

As with preclinical research on sex differences in relation to ethanol drinking, there is also a paucity of preclinical research on sex differences in relation to affective behavior. In regard to exploratory and anxiety-like behavior, most research has been conducted in Wistar and Sprague-Dawley rats, and has found that females compared to males show greater horizontal and vertical (rearing) activity in a novel chamber (Hughes and Hancock, 2016, Kokras et al., 2020), greater or no difference in time in the light chamber of a light-dark box (De Oliveira Sergio et al., 2021, Fleming et al., 2019, Kokras et al., 2020), and more hole exploration in a hole board apparatus (Kokras et al., 2020, Ray and Hansen, 2004). One study has probed the sex-dependent effects of ethanol on behavior in a light-dark box, using experimenter-administered ethanol, and found that this ethanol reduced to an equal extent time spent in the light chamber, entries into the light chamber, and overall ambulatory activity in male and female rats (Fleming et al., 2019). To our knowledge, no such investigations of sex-dependent effects of ethanol have been made of behavior in a novel chamber or hole board apparatus nor has exploratory behavior after voluntary ethanol drinking been examined.

The goal of this study was to examine sex-related differences in patterns of intermittent-access ethanol drinking and their effects on affective behavior. In particular, we wanted to know if and when interventions could be comparable between male and female rats, relative to the number of weeks of ethanol access and to the start of daily ethanol access. Using adult male and female Long-Evans rats with access to ethanol under the 20% ethanol intermittent-access two-bottle-choice paradigm, we compared patterns of drinking across weeks and across a single day of ethanol access. We also compared the effects of this drinking on blood ethanol concentration (BEC) and exploratory and anxiety-like behavior in a novel activity chamber, light-dark box, and hole board apparatus. We hypothesized that, compared to males, female rats would drink more ethanol, possibly with a different 24-hour pattern, but would show similar resulting BECs and similar or greater changes in affective behavior.

MATERIALS AND METHODS

Subjects

Adult, male and female Long-Evans rats (N = 78, n = 39/sex; 7 weeks on arrival at the facility, Charles River Laboratories International, Inc., Malvern, PA, USA) were individually housed in an AAALAC-accredited facility, on a 12-hour reversed light/dark cycle (lights off at 0900 h). They were given one week to acclimate to the facility. All animals received ad libitum chow (Laboratory Rodent Diet 5001, Lab Diet, St. Louis, MO, USA) and water throughout the study. Estrous cycle was not assessed, in order to minimize stress for the females and to reduce the chances of pseudo-pregnancy (Lovick and Zangrossi, 2021, Singletary et al., 2005), and to make as similar as possible the treatment of males and females. Meta-analyses have shown that data collected from female rats does not vary more than data from male rats (Shansky, 2019). Experiments were approved by the Institutional Animal Care and Use Committee of Drexel University College of Medicine and followed the NIH Guide for the Care and Use of Laboratory Animals.

Experimental Protocols

The experimental timeline and usage of animals is schematically represented in Figure 1.

Fig 1.

Fig 1.

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Timeline of experiments and usage of animals. A total of 78 rats were used in this study (n = 39/sex). Of these, 60 (n = 30/sex) were given access to 20% ethanol under the intermittent-access two-bottle-choice paradigm and 18 (n = 9/sex) were given only water and chow. Of the ethanol-drinking rats, 19 (n = 9 males, 10 females) continued past 7 weeks of drinking in this study, and were monitored for their 24-hour pattern of drinking during their 8th week of ethanol access, for affective behavior in their 9th week of access, and for blood ethanol concentration in their 10th week of access. Created with BioRender.com. Abbreviations: BEC, blood ethanol concentration; LDB, light-dark box.

Experiment 1:

To examine possible sex-related differences in weekly drinking levels and patterns under the 20% ethanol intermittent-access two-bottle-choice paradigm, male and female rats (N = 60, n = 30/sex) were given access to ethanol under this paradigm, and their ethanol and water intake was measured daily for 7 weeks (see Ethanol Drinking). To ensure replicability of results, rats were run in three cohorts of approximately 20 rats each (n = ~10/sex). Those not subsequently tested in Experiments 2 and 3 were used in other assays not included in this report. Average drinking was not significantly different between those males and females included in Experiments 2 and 3 and those not included in Experiments 2 and 3 (males: [t(28) = 1.32, p = 0.098]; females: [t(28) = 0.36, p = 0.972]).

Experiment 2:

To determine possible sex-related differences in drinking patterns across a single day of access under the 20% ethanol intermittent-access two-bottle-choice paradigm, some of the rats from Experiment 1 (N = 19, n = 9 males, 10 females), during the second access day of their 8th week of drinking, were monitored for 24 hours. Their ethanol and water intake was measured every half an hour over the first 2 hours and then every 2 hours for the remaining 22 hours. During this time, ethanol and water bottles were also placed on an empty cage and were measured in parallel with those given to the rats, and spillage (determined to be ~0.5 ml per measurement) was accounted for in the analysis of the ethanol and water intake of these 19 rats. Ethanol and water intake was measured by briefly removing and weighing the containers, one subject at a time. To determine BEC, trunk blood was obtained 30 – 40 minutes after the start of daily ethanol access at the termination of the experiment (access week 10) and the BEC was analyzed from frozen plasma with an Analox AM1 Alcohol Analyzer (Lunenburg, MA, USA).

Experiment 3:

To determine possible sex-related differences in the effects of ethanol drinking on exploratory and anxiety-like behavior, the rats from Experiment 2 (N = 19, n = 9 males, 10 females), along with ethanol-naive water drinking control rats (N = 18, n = 9/sex), were given behavioral tests during the 9th week of drinking. The water drinking control rats were run in the same way as the ethanol-drinking rats (ie. one week for acclimation plus 8 weeks with two bottles of water measured daily). The ethanol-drinking rats continued to be given intermittent-access to ethanol during this testing week, with scheduled ethanol given following the conclusion of a behavioral test. On the first day (Monday), rats were tested for activity in a novel chamber; on the second day (Tuesday), they were acclimated to a light-dark box; on the third day (Wednesday), they were tested for behavior in the light-dark box; and on the fifth day (Friday), they were tested for behavior in a hole board apparatus (see Behavioral Testing). Data from one ethanol-drinking male was lost for the activity in a novel chamber test.

Ethanol Drinking

Under the intermittent-access two-bottle-choice paradigm adapted from Wise (1973) and Simms (2008), rats were given unsweetened 20% v/v ethanol during three 24-hour-sessions per week in addition to ad libitum water and chow as described (Barson et al., 2015, Gargiulo et al., 2021). Each Monday, Wednesday, and Friday, one-and-a-half hours after dark onset, one of their two 16 oz (~473 ml) Macrolon bottles of water (Ancare, Bellmore, NY, USA) was replaced with a 16 oz (~473 ml) bottle of ethanol (Ancare) and, after 24 hours, the ethanol was replaced with the second bottle of water. Relative bottle position was alternated each time to prevent side preference and all bottles were fitted with stainless steel ball-bearing non-drip sipper tubes of equal size. Ethanol intake was calculated as: (weight ethanol solution consumed (g) * (density ethanol * 0.20)) / rat body weight (kg). Ethanol preference was calculated as: volume ethanol solution consumed (ml) / (volume ethanol solution consumed (ml) + volume water consumed (ml)). Animals were weighed on Tuesdays and Fridays. Animals in the water control group were treated exactly as those in the ethanol group, but had two bottles of water at all times.

Behavioral Testing

All behavioral testing was conducted in a sound- and light-attenuated room (< 5 lux, or approximately as bright as moonlight), starting one-and-a-half hours into the dark cycle, at a time when animals would normally receive ethanol (24 or 48 hours into abstinence). Ethanol-trained rats were given access to their scheduled ethanol following the conclusion of the behavioral test, once they were returned to their vivarium room. During transport between rooms, cages were fully covered with an opaque drape. All rats were acclimated to the testing room for 5 minutes prior to each test.

Activity in a Novel Chamber:

Locomotor activity was assessed in an automated activity chamber with an area of 43.2 cm × 43.2 cm and 42 cm high walls (Med Associates, Inc., St. Albans, VT, USA). Animals with no prior experience in this chamber were placed at the center of the chamber and allowed to explore for 15 minutes, while ambulatory time and distance and vertical time (rearing) were measured via infrared beams (Barson et al., 2015, Pirino et al., 2020).

Light-Dark Box:

A light/dark insert (Med Associates, Inc., St. Albans, VT, USA) was placed in the same chamber used for activity testing, to create a two-chamber light-dark box. A lamp was placed directly above the light chamber, creating a luminous intensity of approximately 400 lux in that chamber. Animals were placed in the light chamber and allowed to explore the light-dark box for 5 minutes. First, they were given one 5-minute period for acclimation to the chamber, as we and others have found that behavior during the first time in the light-dark box is different from behavior during subsequent times, with behavior during the subsequent times showing reduced locomotor activity and increased time in the light chamber (Bouwknecht et al., 2004, Rodgers and Shepherd, 1993). The next day, the rats were tested in the same chamber for 5 minutes. During both 5-minute periods, time spent in the light chamber, number of entries into the two chambers, and ambulatory time were measured via infrared beams (Barson et al., 2015, Gargiulo et al., 2021).

Hole Board Apparatus:

A hole-board insert (Med Associates, Inc., St. Albans, VT, USA), comprised of 16 equidistant receptacle holes (3 cm in diameter) on a 43 cm × 43 cm metal platform, was placed in the same chamber used for locomotor activity testing. The hole-board stood 5 cm above the floor of the chamber and was not baited. Each animal was placed in the corner of the chamber and allowed to explore for 5 minutes, while nose pokes into novel, repeat, and total holes were recorded via infrared beams (Pandey et al., 2019, Pandey and Barson, 2020).

Data Analysis

Sphericity of the data was determined using Mauchly’s test, and a Greenhouse-Geisser correction was used when sphericity was violated. When this correction was applied, the degrees of freedom were changed by the correction. To compare weekly ethanol and water drinking and weekly ethanol preference between male and female rats and across weeks, a mixed ANOVA was used, with average weekly intake or preference as the within-subject factor and sex as the between-subject factor. To compare ethanol drinking at timepoints across a single day, a mixed ANOVA was used for the 2-hour measurements and the 30-minute measurements, with time as the within-subject factor and sex as the between-subject factor. Significant main and interaction effects were followed up with Sidak pairwise comparison tests. To identify sub-groups of ethanol drinkers, based on their weekly ethanol drinking and also their drinking across a single day, a two-step cluster analysis was used and was followed up with a k-means cluster analysis. To compare BECs and ethanol, water, and food intake between male and female rats, independent-samples two-tailed t-tests were used. To determine if ethanol intake predicted BEC and exploratory behaviors, linear regression was used. To compare behaviors between ethanol and water drinkers and between male and female rats, two-way ANOVAs were used, with substance and sex as between-subject factors. All data were analyzed using SPSS (Version 28, IBM, Armonk, NY, USA). Significance was determined at p < 0.05 and all p values are reported with up to three significant figures. Data are reported as mean ± standard error of the mean (S.E.M.).

RESULTS

Experiment 1: Sex-related differences in level and pattern of weekly ethanol drinking and preference

To determine possible sex-related differences in weekly drinking levels and patterns under the 20% ethanol intermittent-access two-bottle-choice paradigm, male and female rats (N = 60, n = 30/sex) were given access to ethanol under this paradigm, and their daily intake was measured for 7 weeks. Across these 7 weeks, male rats averaged 6.71 ± 0.45 g/kg/d ethanol intake and females averaged nearly double this intake, at 13.13 ± 0.73 g/kg/d ethanol. A mixed ANOVA for average weekly ethanol intake revealed that there were significant main effects of both sex [F(1, 58) = 56.37, p < 0.001] and week [F(4.43, 257.10) = 3.11, p = 0.013], and a significant interaction between sex and week [F(4.43, 257.10) = 2.48, p = 0.039] (Figure 2A). Females drank significantly more ethanol than males, and this occurred at each of the 7 access weeks (p = 0.002 - p < 0.001). Weekly intake for males was stable beginning at access week 1, being not significantly different between any of the 7 weeks (p = 0.675 – p = 1.000). In contrast, weekly intake for females was not stable until access week 5, with week 2 being significantly different from weeks 3 and 5 (p = 0.019 and p < 0.001, respectively) and week 4 also being significantly different from week 5 (p = 0.025). Drinking in week 1 and week 7 was not significantly different for males (p = 0.994) or females (p = 1.000). A mixed ANOVA for average weekly water intake revealed only a significant main effect of sex [F(1, 58) = 38.79, p < 0.001], with males drinking significantly more water than females. For ethanol preference, male rats escalated from 25.27% to 37.34% while females escalated from 36.33% to 52.54% across the 7 weeks of ethanol access. As with average weekly ethanol intake, a mixed ANOVA for average ethanol preference revealed that there were significant main effects of both sex [F(1, 58) = 21.04, p < 0.001] and week [F(4.00, 232.04) = 26.70, p < 0.001], and a significant interaction between sex and week [F(4.00, 232.04) = 3.43, p = 0.01] (Figure 2B). Females had a significantly higher ethanol preference than males, and this occurred at each of the 7 weeks (p = 0.022 – p < 0.001). In contrast to ethanol intake, however, ethanol preference for males was not stable until access week 4, with weeks 1 – 3 being significantly different from all subsequent weeks (p = 0.014 – p < 0.001). Similar to weekly ethanol intake, weekly ethanol preference for females was not stable until access week 5, with weeks 1 – 4 being significantly different from all subsequent weeks (p = 0.039 – p < 0.001). Preference in week 7 was significantly greater than in week 1 for both males (p < 0.001) and females (p < 0.001). Together, these results demonstrate that females have higher ethanol intake and preference than males across 7 weeks of ethanol access, but that the pattern of intake and preference across weeks varies between the sexes, with both of these measures becoming stable for both males and females by week 5 of access.

Fig 2.

Fig 2.

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Ethanol intake and preference in male and female rats (N = 60, n = 30/sex) across 7 weeks of drinking under the 20% ethanol intermittent-access two-bottle-choice paradigm (Experiment 1). A. Average ethanol intake (reported as pure ethanol in grams per rat body weight in kilograms) was significantly greater in female compared to male rats across all 7 weeks of drinking. B. Average ethanol preference increased across weeks in both males and females but was significantly greater in female compared to male rats across all 7 weeks of drinking. C. Cluster analysis of average ethanol intake revealed two distinct groups of ethanol drinkers, with the low drinkers consisting of all of the male rats (n = 30) and about half (n = 14) of the female rats, and the high drinkers consisting of the other half (n = 16) of the female rats. D. Break-down of average ethanol intake of the male rats (n = 30), low-drinking female rats (n = 14), and high-drinking female rats (n = 16). Data are mean ± S.E.M., **p < 0.01, *p < 0.05 vs. males.

Cluster analysis of average weekly ethanol intake identified two groups of drinkers. Animals in the first group drank lower levels of ethanol, averaging 6.63 ± 0.40 g/kg/d ethanol intake and consisting of all of the male rats (n = 30) and about half (n = 14) of the female rats. Animals in the second group drank higher levels of ethanol, averaging 16.20 ± 0.63 g/kg/d ethanol intake and consisting of the other half (n = 16) of the female rats. A mixed ANOVA for average weekly ethanol intake confirmed that there was a significant main effect of group [F(1, 58) = 126.83, p < 0.001] and revealed that there was also a significant main effect of week [F(4.43, 257.47) = 3.08, p = 0.014], but not a significant interaction between group and week [F(4.44, 257.47) = 0.80, p = 0.535] (Figure 2C). Thus, while female rats as a group have higher ethanol intake than males, this difference is driven by about half of the females, while the other half drink at levels more similar to males (Figure 2D).

Experiment 2: Comparison between the sexes in drinking pattern across a single day of ethanol access

To determine possible sex-related differences in drinking patterns across a single day of access under the 20% ethanol intermittent-access two-bottle-choice paradigm, male and female rats (N = 19, n = 9 males, 10 females), during their 8th week of drinking, were monitored for 24 hours. A mixed ANOVA for 2-hour ethanol intake across 24 hours revealed that there were significant main effects of both sex [F(1, 17) = 21.72, p < 0.001] and time [F(4.27, 72.56) = 27.81, p < 0.001], and a significant interaction between sex and time [F(4.27, 72.56) = 3.03, p = 0.021] (Figure 3A). Females drank significantly more ethanol than males, and this occurred at each measurement point (p = 0.034 – p < 0.001) except hour 18 (p = 0.066). While the level of intake varied for both sexes across time, intake for both groups was lower than other timepoints during hours 12 – 14, 14 – 16, and 16 – 18 (p = 0.045 – p < 0.001), the middle of the light phase of the light/dark cycle. The highest numerical measurement points for both sexes occurred during the access periods of 0 – 2 and 22 – 24 hours, but 0 – 2 hours for males was not significantly different than 22 – 24 hours (1.45 ± 0.17 vs. 0.90 ± 0.14 g/kg/2 hr, p = 0.977), while 0 – 2 hours in females was significantly greater than 22 – 24 hours (3.02 ± 0.28 vs. 1.66 ± 0.12 g/kg/2 hr, p = 0.003). Similarly, between the periods of 0 – 2 and 2 – 4, intake dropped 57% in males and 54% in females; in contrast, between the periods of 20 – 22 and 22 – 24, intake increased 52% in males but only 37% in females. A mixed ANOVA for 30-minute ethanol intake across the first 2 hours of access also revealed that there were significant main effects of both sex [F(1, 17) = 22.39, p < 0.001] and time [F(2.21, 37.53) = 21.04, p < 0.001], but the interaction between sex and time showed only a trend for significance [F(2.21, 37.53) = 2.83, p = 0.067] (Figure 3B). With males and females analyzed as a single group, intake was highest in the first 30 minutes of access, compared to all subsequent 30-minute periods across the next 90 minutes (p = 0.004 – p < 0.001). Thus, peak ethanol intake for both males and females appears to be in the first 30 minutes of daily access.

Fig 3.

Fig 3.

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Ethanol intake across a 24-hour period of 20% ethanol access in male and female rats (N = 19, n = 9 males, 10 females) under the intermittent-access two-bottle-choice paradigm (Experiment 2). A. When measured every 2 hours, females drank more ethanol (reported as pure ethanol in grams per rat body weight in kilograms) than males at almost every time-point, but followed the same general pattern, with the highest intake during the first measurement period and the lowest intake during the middle of the light phase of the light/dark cycle. B. When measured every 30 minutes, both males and females drank the most ethanol during first 30 minutes of access, compared to all subsequent periods across the first 2 hours of access. C. Cluster analysis of 2-hour ethanol intake revealed two distinct groups of ethanol drinkers, with the low drinkers consisting of all but one of the male rats (n = 8) and about half (n = 4) of the female rats, and the high drinkers consisting of the other half (n = 6) of the female rats along with one of the male rats. D. Break-down of average ethanol intake of the low-drinking male rats (n = 8), low-drinking female rats (n = 4), and high-drinking female rats (n = 6). Data are mean ± S.E.M., **p < 0.01, *p < 0.05 vs. males.

Cluster analysis of 2-hour ethanol intake again identified two groups of drinkers. Animals in the first group drank lower levels of ethanol (8.27 ± 0.86 g/kg/d) and consisted of all but one of the male rats (n = 8) and about half (n = 4) of the female rats. Animals in the second group drank higher levels of ethanol (19.39 ± 1.13 g/kg/d) and consisted of the other half (n = 6) of the female rats along with one of the male rats. A mixed ANOVA for 2-hour ethanol intake across 24 hours confirmed that there was a significant main effect of group [F(1, 17) = 59.65, p < 0.001] and revealed that there was a significant main effect of time [F(4.15, 70.48) = 29.12, p < 0.001], but the interaction between group and time showed only a trend for significance [F(4.15, 70.48) = 2.18, p = 0.078] (Figure 3C). These results suggest that, similar to weekly intake, female rats as a group have higher ethanol intake than males across 24 hours of daily access, but that this difference is largely driven by about half of the females, and that they drink in the same overall daily pattern as the lower-drinking males (Figure 3D).

With blood obtained 30 – 40 minutes after the start of daily ethanol access, BEC levels for males ranged from 1 – 83 mg/dl and those for females ranged from 0 – 136 mg/dl. Despite a significantly greater 30-minute ethanol intake by female rats [1.34 ± 0.15 vs. 0.60 ± 0.16 g/kg/30 min; t(17) = −3.13, p = 0.006], BECs were not significantly different between males and females [t(17) = −0.10, p = 0.333]. On the other hand, BECs were significantly predicted by ethanol intake (R2 = 0.61, p < 0.001). While there was no significant difference between males and females for 30-minute water intake [t(17) = 1.22, p = 0.239], females ate significantly less chow than males during this time [t(17) = 2.29, p = 0.035]. These results suggest that, despite higher levels of ethanol intake by females, resulting BECs are not significantly different between male and female rats.

Experiment 3: Comparison between the sexes in effects of ethanol drinking on affective behavior

To determine possible sex-related differences in the effects of ethanol drinking on exploratory and anxiety-like behavior, male and female rats (N = 19, n = 9 males, 10 females), along with ethanol-naive water drinking control rats (N = 18, n = 9/sex), were given behavioral tests during the 9th week of drinking, at a time when ethanol-trained rats would normally receive ethanol. For exploratory activity in a novel chamber, a two-way ANOVA revealed no significant main effects of sex or ethanol on ambulatory time (sex: [F(1, 32) = 0.43, p = 0.517]; ethanol: [F(1, 32) = 0.83, p = 0.368]) (Figure 4A) or ambulatory distance (sex: [F(1, 32) = 1.99, p = 0.168]; ethanol: [F(1, 32) = 1.54, p = 0.224]) (Figure 4B), but males showed significantly greater vertical time than females [F(1, 32) = 9.54, p = 0.004], and ethanol compared to water led to a trend for reduced vertical time [F(1, 32) = 3.56, p = 0.068] (Figure 4C). Among ethanol drinkers, average weekly ethanol drinking significantly predicted both ambulatory time and ambulatory distance for females (R2 = 0.40, p = 0.048; R2 = 0.46, p = 0.038, respectively), but did not predict any behavior in the activity chamber for males (R2 = 0.00 – 0.11, p = 0.422 – 0.954) (Figures 4D – F). In the females, higher ethanol drinking predicted lower ambulatory time and ambulatory distance.

Fig 4.

Fig 4.

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Locomotor activity in a novel chamber in male and female rats with (N = 19, n = 8 males, 10 females) and without (N = 18, n = 9/sex) a history of ethanol drinking (Experiment 3). A. Ambulatory time was not significantly different between males and females or between ethanol and water drinkers. B. Ambulatory distance was not significantly different between males and females or between ethanol and water drinkers. C. Males showed significantly more vertical time (rearing behavior) than females. D. Among ethanol drinkers, average weekly ethanol drinking significantly predicted ambulatory time for females but not males. E. Among ethanol drinkers, average weekly ethanol drinking significantly predicted ambulatory distance for females but not males. F. Among ethanol drinkers, average weekly ethanol drinking did not significantly predict vertical time for males or females. Data are mean ± S.E.M., **p < 0.01 vs. males.

For exploratory and anxiety-like behavior in a light-dark box, a two-way ANOVA of behavior during the second exposure (the test) revealed no significant main effects of sex or ethanol on time in the light chamber (sex: [F(1, 33) = 0.01, p = 0.920]; ethanol: [F(1, 33) = 2.33, p = 0.137]) (Figure 5A) or total ambulatory time (sex: [F(1, 33) = 2.23, p = 0.145]; ethanol: [F(1, 33) = 0.70, p = 0.409]) (Figure 5C), but females showed significantly more movement between the chambers (“zone entries”) than males [F(1, 33) = 5.33, p = 0.027], and ethanol led to a trend for increased zone entries [F(1, 33) = 3.56, p = 0.068] (Figure 5B). Similar to these results, a two-way ANOVA of behavior during the first exposure (the acclimation) revealed no significant main effects of sex or ethanol on time in the light chamber (sex: [F(1, 33) = 0.25, p = 0.620]; ethanol: [F(1, 33) = 0.02, p = 0.896]) or total ambulatory time (sex: [F(1, 33) = 0.52, p = 0.476]; ethanol: [F(1, 33) = 1.59, p = 0.216]), but females again showed significantly more movement between the chambers than males [F(1, 33) = 7.55, p = 0.010], and ethanol again led to a trend for increased zone entries [F(1, 33) = 3.16, p = 0.085]. Among ethanol drinkers, average weekly ethanol drinking again significantly predicted ambulatory time for females (R2 = 0.41, p = 0.046) and it predicted both ambulatory time (R2 = 0.67, p = 0.007) and time in the light chamber (R2 = 0.50, p = 0.032) for males, during the second exposure (Figures 5D – F). In the females, higher ethanol drinking predicted lower ambulatory time, while in the males, it instead predicted higher ambulatory time and time in the light chamber.

Fig 5.

Fig 5.

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Exploration and anxiety-like behavior in a light-dark box in male and female rats with (N = 19, n = 9 males, 10 females) and without (N = 18, n = 9/sex) a history of ethanol drinking (Experiment 3). A. Time spent in the light chamber was not significantly different between males and females or between ethanol and water drinkers. B. Females showed significantly more movement between the chambers (“zone entries”) than males. C. Ambulatory time was not significantly different between males and females or between ethanol and water drinkers. D. Among ethanol drinkers, average weekly ethanol drinking significantly predicted time in the light chamber for males but not females. E. Among ethanol drinkers, average weekly ethanol drinking did not significantly predict movement between the chambers for males or females. F. Among ethanol drinkers, average weekly ethanol drinking significantly predicted ambulatory time for both males and females. Data are mean ± S.E.M., *p < 0.05 vs. males.

For novelty-seeking, exploratory behavior in a hole board apparatus, a two-way ANOVA revealed that females made significantly more nose pokes than males into novel holes [F(1, 33) = 5.26, p = 0.028], with no significant main effects of ethanol on this behavior [F(1, 33) = 0.43, p = 0.836] (Figure 6A), and while there were no significant main effects of sex or ethanol on repeat nose pokes into the same holes (sex: [F(1, 33) = 3.00, p = 0.092]; ethanol: [F(1, 33) = 0.02, p = 0.877]) (Figure 6B), females nevertheless showed a strong trend for making more total hole pokes than males [F(1, 33) = 3.99, p = 0.054] (Figure 6C). Average weekly ethanol drinking did not significantly predict any behavior in this test in either males or females (Figures 6D – F). Altogether, the data suggest that males engage in more vertical exploratory behavior while females engage in more horizontal exploratory behavior, and that while ethanol history may alter these behaviors, it does not do so in a clear sex-dependent manner.

Fig 6.

Fig 6.

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Novelty-seeking, exploratory behavior in a hole board apparatus in male and female rats with (N = 19, n = 9 males, 10 females) and without (N = 18, n = 9/sex) a history of ethanol drinking (Experiment 3). A. Females made significantly more nose pokes than males into novel holes. B. Nose pokes into the same (repeat) holes were not significantly different between males and females or between ethanol and water drinkers. C. Females showed a strong trend for more total nose pokes than males. D. Among ethanol drinkers, average weekly ethanol drinking did not significantly predict nose pokes into novel holes for males or females. E. Among ethanol drinkers, average weekly ethanol drinking did not significantly predict nose pokes into the same holes for males or females. F. Among ethanol drinkers, average weekly ethanol drinking did not significantly predict total nose pokes for males or females. Data are mean ± S.E.M., *p < 0.05, #p < 0.06 vs. males.

DISCUSSION

In this study, we found that adult female Long-Evans rats as a group showed higher levels of ethanol intake and preference than males under the 20% ethanol intermittent-access two-bottle-choice paradigm, although their intake and preference took longer to stabilize. Over the course of a day, males and females showed a similar pattern of ethanol drinking. About half of females drank significantly higher levels of ethanol than males, while the other half drank at levels more similar to males. Despite their higher overall levels of intake, females did not show significant differences from males in their BEC levels. Further, while there were sex-related differences in affective behavior, they did not show a significant interaction with ethanol drinking. These results together highlight that, depending on the experiment, different considerations may need to be made for female compared to male rats when designing studies involving intermittent access ethanol drinking. We suggest, for example, that female and male rats could both be tested for acute effects of ethanol after 30 minutes of daily access and 5 total weeks of access, but that BECs may be a better way to determine equivalent drinking between males and females than actual ethanol intake. Moreover, since sex and level of intake appear to be partially segregated as factors, this should also be taken into consideration when designing studies involving ethanol drinking. For example, for scientists looking to include only the highest-drinking animals, experiments should predominantly include female animals. In contrast, for scientists looking to have representation of high-drinking male and female animals, experiments should first segregate animals by sex. Overall, there are multiple factors to consider, depending on the ultimate aims of any ethanol study.

A major finding of these experiments is that, while females showed higher levels of ethanol intake and preference than males across all 7 weeks of drinking, male intake was stable throughout all weeks of access and male preference reached stability by week 4, while female intake and preference did not become stable until week 5 of access. While it cannot be ruled out that this delay in stabilization in female drinking could be attributable to estrous cycle fluctuations, we believe this is unlikely, in light of prior findings that intermittent-access ethanol drinking is not dependent on estrous cycle stage (Li et al., 2019, Priddy et al., 2017) and that our subjects were run in three separate cohorts. While a number of other studies that examined intermittent-access ethanol-drinking in rats have similarly found higher levels of drinking and preference in females compared to males (Li et al., 2019, Loi et al., 2014, Priddy et al., 2017, McNamara and Ito, 2021), others have reported no difference (Quadir et al., 2022, Schramm-Sapyta et al., 2014). Of the two published studies that specifically examined Long-Evans rats drinking 20% ethanol, one reported that females drank more than males over 5 weeks of access (McNamara and Ito, 2021), while the other found that females drank more over the first 3 weeks of access, but not during subsequent weeks (Morales et al., 2015). The apparent contradiction between this later study and the present results (as well as the former study) may be due to differences in vendor (Harlan vs. Charles River) or laboratory, as the drinking levels in both sexes in the later study averaged around 5 g/kg/day, which is more similar to the male drinking levels in the present experiment (6.71 g/kg/day) and the former study. Of note, cluster analysis of the present results demonstrated that it was roughly half of the females, rather than all, that drank higher levels of ethanol than males, not unlike the different phenotypes of male intermittent-access drinkers identified in earlier studies (Ehinger et al., 2021, Pandey and Barson, 2020). Examining weekly levels of intermittent-access 20% ethanol drinking, different studies have reported different patterns. While some have found escalating levels of intake across weeks in both males and females (Loi et al., 2014, Schramm-Sapyta et al., 2014), others have found it in males but not females (Morales et al., 2015) or in neither males nor females (Quadir et al., 2022). In our own work with males, we have previously observed both stable intake across all access weeks and escalation until week 3 or 4 of access (Barson et al., 2015, Pandey et al., 2019), so part of this measure may depend on other experimental conditions. Interestingly, both males and females in the present study demonstrated an escalation in ethanol preference across all weeks of access, suggesting that changes in ethanol intake were not due to changes in fluid intake alone. Assessed by either intake or preference, however, the results of the present study indicate that female Long-Evans rats engage with ethanol more than males over multiple weeks of access under the 20% ethanol intermittent-access model.

Over an ethanol access day, the females and males with a history of ethanol drinking consumed ethanol in roughly the same pattern, and this led to similar BECs. Their peak time of intake occurred in the first two hours of daily access, specifically in the first 30 minutes of access, and they had a second peak during the final two hours of daily access, while their lowest intake occurred in the middle of the light phase of the light/dark cycle. To our knowledge, this is the first time that the 24-hour intake of male or female rats has been described for this access model. Prior research has found that rats drink a high percentage of their daily intake in the first 30 minutes of access (Quadir et al., 2022, Barson et al., 2015) and that most consumption occurs during the dark cycle (Bell et al., 2011). Similarly, across a 24-hour period in a 15-day on, 15-day off intermittent-access model, male and female P and HAD2 rats were found to follow a circadian pattern of ethanol intake, although this was not statistically compared between the sexes (Rosenwasser et al., 2014). The finding of similar BECs despite higher ethanol intake in females is in line with results from several other studies (Kulkosky, 1980, Lancaster and Spiegel, 1992) and is consistent with work showing that females compared to males have reduced BECs following the same experimenter-administered dose of ethanol (Mankes et al., 1991). Thus, while the present results are in line with published research, they confirm for the first time that assays probing the direct effects of ethanol drinking could be performed at the end of 30 minutes of access in both male and female Long-Evans rats.

In regard to affective behavior, several sex-related differences were identified and, following 8 weeks of drinking under the intermittent-access model, there were several trends for effects of ethanol, but no significant interactions between sex and ethanol. Compared to males, females showed less time vertical in a novel activity chamber, more movement between chambers in a light-dark box, and more nose pokes in a hole board apparatus. Compared to water-drinking control rats, those with a history of ethanol drinking showed a trend for reduced vertical time in the novel activity chamber and more zone entries in the light-dark box. Thus, in the present experiment, while males appeared to engage in more vertical exploratory behavior, females engaged in more horizontal exploratory behavior, and a history of ethanol drinking somewhat altered behavior in a manner more consistent with the female phenotype. While prior research using both Wistar and Long-Evans rats has reported that females show greater horizontal and vertical (rearing) behavior in a novel chamber (Hughes and Hancock, 2016, Kokras et al., 2020), these tests were run in the light phase of the light/dark cycle, when rats are normally more likely to sleep. With the present study run during the dark cycle, it is possible that the timing of the testing explains the discrepancy in results. For the light-dark box, prior research has examined Wistar and Sprague-Dawley rats and found that females either show more or the same amount of time in the light chamber and transitions between the chambers (De Oliveira Sergio et al., 2021, Fleming et al., 2019, Kokras et al., 2020, Viviani et al., 2012). By examining Long-Evans rats, the present research adds another strain of rat to these findings. For the hole-board apparatus, prior research has examined Wistar rats and has been consistent in finding that females explore more holes than males (Kokras et al., 2020, Ray and Hansen, 2004), just as was found for Long-Evans rats in the current study. The finding that a history of ethanol drinking somewhat reduced vertical time in a novel activity chamber and increased zone entries in a light-dark box is inconsistent with the very limited prior research, which found that ad libitum ethanol drinking increased vertical time in a novel chamber in male alcohol-preferring P rats (Hauser et al., 2020) and that experimenter-administered ethanol led to reduced time spent in and number of entries into the light chamber of a light-dark box in male and female Sprague-Dawley rats (Fleming et al., 2019). On the other hand, there are major differences between the ethanol exposure paradigms and rat strains that could easily explain the differences in these results. Interestingly, among ethanol drinking females in the present study, a history of higher ethanol drinking predicted lower ambulatory time and distance in the novel activity chamber and light-dark box, whereas among males, higher drinking predicted higher ambulatory time and time in the light chamber of the light-dark box. This suggests that there may be subtle sex-dependent effects of a history of ethanol drinking that were masked in the ANOVA, and that there may have been a Type II statistical error that would not have occurred with the inclusion of more subjects. Moreover, it could inform on the use of the high responder / low responder model, which identifies animals with increased exploratory behavior in a novel environment that are found to be more prone to engaging with drugs of abuse (Blanchard et al., 2009, Flagel et al., 2014). Specifically, the current results suggest that this model may be more appropriate for males than for females in relation to intermittent access ethanol drinking. Overall however, the present results demonstrate that, despite their baseline differences in affective behavior and levels of ethanol drinking, male and female rats do not show significantly different effects of intermittent-access ethanol drinking on exploratory and anxiety-like behavior.

In summary, this study describes for the first time the patterns of intermittent-access ethanol drinking in male and female rats, both across weeks and across a single day of drinking, and their impact on exploratory and anxiety-like behavior. It identifies sex-related differences in levels of ethanol intake and preference and their time to reach stability, but an overall similarity in drinking pattern over the course of 24 hours of daily access. Moreover, while it identifies sex-related differences in baseline exploratory behavior, it finds that the overall effects of a history of ethanol drinking do not appear to be sex-dependent. In identifying where male and female rats do and do not differ in their ethanol drinking patterns and their subsequent effects on behavior, the results of this study emphasize points at which different considerations should be made for female and male rats when designing studies that involve intermittent-access ethanol drinking.

ACKNOWLEDGMENTS

This research was supported by the National Institute on Alcohol Abuse and Alcoholism under Award Numbers R01AA028228 (J.R.B. and A.N.K.), R01AA028218 (J.R.B.), and R21AA027629 (J.M.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors declare no conflict of interest.

REFERENCES

  1. Barson JR, Ho HT & Leibowitz SF 2015. Anterior thalamic paraventricular nucleus is involved in intermittent access ethanol drinking: role of orexin receptor 2. Addict Biol, 20: 469–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell RL, Rodd ZA, Smith RJ, Toalston JE, Franklin KM & Mcbride WJ 2011. Modeling binge-like ethanol drinking by peri-adolescent and adult P rats. Pharmacol Biochem Behav, 100: 90–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blanchard MM, Mendelsohn D & Stamp JA 2009. The HR/LR model: Further evidence as an animal model of sensation seeking. Neurosci Biobehav Rev, 33: 1145–54. [DOI] [PubMed] [Google Scholar]
  4. Bouwknecht JA, Van Der Gugten J, Groenink L, Olivier B & Paylor RE 2004. Effects of repeated testing in two inbred strains on flesinoxan dose-response curves in three mouse models for anxiety. Eur J Pharmacol, 494: 35–44. [DOI] [PubMed] [Google Scholar]
  5. Darevsky D, Gill TM, Vitale KR, Hu B, Wegner SA & Hopf FW 2019. Drinking despite adversity: behavioral evidence for a head down and push strategy of conflict-resistant alcohol drinking in rats. Addict Biol, 24: 426–437. [DOI] [PubMed] [Google Scholar]
  6. De Oliveira Sergio T, Lei K, Kwok C, Ghotra S, Wegner SA, Walsh M, Waal J, Darevsky D & Hopf FW 2021. The role of anterior insula-brainstem projections and alpha-1 noradrenergic receptors for compulsion-like and alcohol-only drinking. Neuropsychopharmacology, 46: 1918–1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ehinger Y, Phamluong K, Darevesky D, Welman M, Moffat JJ, Sakhai SA, Whiteley EL, Berger AL, Laguesse S, Farokhnia M, Leggio L, Lordkipanidze M & Ron D 2021. Differential correlation of serum BDNF and microRNA content in rats with rapid or late onset of heavy alcohol use. Addict Biol, 26: e12890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eriksson K 1972. Behavioral and physiological differences among rat strains specially selected for their alcohol consumption. Ann N Y Acad Sci, 197: 32–41. [DOI] [PubMed] [Google Scholar]
  9. Flagel SB, Waselus M, Clinton SM, Watson SJ & Akil H 2014. Antecedents and consequences of drug abuse in rats selectively bred for high and low response to novelty. Neuropharmacology, 76 Pt B: 425–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fleming W, Jones Q, Chandra U, Saini A, Walker D, Francis R, Ocampo G & Kuhn C 2019. Withdrawal from brief repeated alcohol treatment in adolescent and adult male and female rats. Alcohol Clin Exp Res, 43: 204–211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gargiulo AT, Pirino BE, Curtis GR & Barson JR 2021. Effects of pituitary adenylate cyclase-activating polypeptide isoforms in nucleus accumbens subregions on ethanol drinking. Addict Biol, 26: e12972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guizzetti M, Davies DL, Egli M, Finn DA, Molina P, Regunathan S, Robinson DL & Sohrabji F 2016. Sex and the lab: An alcohol-focused commentary on the NIH initiative to balance sex in cell and animal studies. Alcohol Clin Exp Res, 40: 1182–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hasin DS, Shmulewitz D & Keyes K 2019. Alcohol use and binge drinking among U.S. men, pregnant and non-pregnant women ages 18–44: 2002–2017. Drug Alcohol Depend, 205: 107590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hauser SR, Smith RJ, Toalston JE, Rodd ZA, Mcbride WJ & Bell RL 2020. Spontaneous early withdrawal behaviors after chronic 24-hour free-choice access to ethanol. Alcohol Alcohol, 55: 480–488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hughes RN & Hancock NJ 2016. Strain-dependent effects of acute caffeine on anxiety-related behavior in PVG/c, Long-Evans and Wistar rats. Pharmacol Biochem Behav, 140: 51–61. [DOI] [PubMed] [Google Scholar]
  16. Kokras N, Dioli C, Paravatou R, Sotiropoulos MG, Delis F, Antoniou K, Calogeropoulou T, Charalampopoulos I, Gravanis A & Dalla C 2020. Psychoactive properties of BNN27, a novel neurosteroid derivate, in male and female rats. Psychopharmacology (Berl), 237: 2435–2449. [DOI] [PubMed] [Google Scholar]
  17. Kulkosky PJ 1980. Blood ethanol and free-choice ethanol intake in rats amd their progeny. Pharmacol Biochem Behav, 13: 449–52. [DOI] [PubMed] [Google Scholar]
  18. Lancaster FE & Spiegel KS 1992. Sex differences in pattern of drinking. Alcohol, 9: 415–20. [DOI] [PubMed] [Google Scholar]
  19. Li J, Chen P, Han X, Zuo W, Mei Q, Bian EY, Umeugo J & Ye J 2019. Differences between male and female rats in alcohol drinking, negative affects and neuronal activity after acute and prolonged abstinence. Int J Physiol Pathophysiol Pharmacol, 11: 163–176. [PMC free article] [PubMed] [Google Scholar]
  20. Loi B, Colombo G, Maccioni P, Carai MA, Franconi F & Gessa GL 2014. High alcohol intake in female Sardinian alcohol-preferring rats. Alcohol, 48: 345–51. [DOI] [PubMed] [Google Scholar]
  21. Lovick TA & Zangrossi H Jr. 2021. Effect of estrous cycle on behavior of females in rodent tests of anxiety. Front Psychiatry, 12: 711065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mankes RF, Glick SD, Van Der Hoeven T & Lefevre R 1991. Alcohol preference and hepatic alcohol dehydrogenase activity in adult Long-Evans rats is affected by intrauterine sibling contiguity. Alcohol Clin Exp Res, 15: 80–5. [DOI] [PubMed] [Google Scholar]
  23. Mcnamara TA & Ito R 2021. Relationship between voluntary ethanol drinking and approach-avoidance biases in the face of motivational conflict: novel sex-dependent associations in rats. Psychopharmacology (Berl), 238: 1817–1832. [DOI] [PubMed] [Google Scholar]
  24. Morales M, Mcginnis MM & Mccool BA 2015. Chronic ethanol exposure increases voluntary home cage intake in adult male, but not female, Long-Evans rats. Pharmacol Biochem Behav, 139: 67–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pandey S, Badve PS, Curtis GR, Leibowitz SF & Barson JR 2019. Neurotensin in the posterior thalamic paraventricular nucleus: inhibitor of pharmacologically relevant ethanol drinking. Addict Biol, 24: 3–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pandey S & Barson JR 2020. Heightened exploratory behavior following chronic excessive ethanol drinking: Mediation by neurotensin receptor type 2 in the anterior paraventricular thalamus. Alcohol Clin Exp Res, 44: 1747–1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pirino BE, Spodnick MB, Gargiulo AT, Curtis GR, Barson JR & Karkhanis AN 2020. Kappa-opioid receptor-dependent changes in dopamine and anxiety-like or approach-avoidance behavior occur differentially across the nucleus accumbens shell rostro-caudal axis. Neuropharmacology, 181: 108341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Priddy BM, Carmack SA, Thomas LC, Vendruscolo JC, Koob GF & Vendruscolo LF 2017. Sex, strain, and estrous cycle influences on alcohol drinking in rats. Pharmacol Biochem Behav, 152: 61–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Quadir SG, Arleth GM, Jahad JV, Echeveste Sanchez M, Effinger DP & Herman MA 2022. Sex differences in affective states and association with voluntary ethanol intake in Sprague-Dawley rats. Psychopharmacology (Berl). [DOI] [PubMed] [Google Scholar]
  30. Ray J & Hansen S 2004. Temperament in the rat: sex differences and hormonal influences on harm avoidance and novelty seeking. Behav Neurosci, 118: 488–97. [DOI] [PubMed] [Google Scholar]
  31. Rodgers RJ & Shepherd JK 1993. Influence of prior maze experience on behaviour and response to diazepam in the elevated plus-maze and light/dark tests of anxiety in mice. Psychopharmacology (Berl), 113: 237–42. [DOI] [PubMed] [Google Scholar]
  32. Rosenwasser AM, Mcculley WD 3rd & Fecteau M 2014. Circadian activity rhythms and voluntary ethanol intake in male and female ethanol-preferring rats: effects of long-term ethanol access. Alcohol, 48: 647–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schramm-Sapyta NL, Francis R, Macdonald A, Keistler C, O’neill L & Kuhn CM 2014. Effect of sex on ethanol consumption and conditioned taste aversion in adolescent and adult rats. Psychopharmacology (Berl), 231: 1831–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shansky RM 2019. Are hormones a “female problem” for animal research? Science, 364: 825–826. [DOI] [PubMed] [Google Scholar]
  35. Shmulewitz D, Aharonovich E, Witkiewitz K, Anton RF, Kranzler HR, Scodes J, Mann KF, Wall MM, Hasin D & Alcohol Clinical Trials I 2021. The World Health Organization risk drinking levels measure of alcohol consumption: Prevalence and health correlates in nationally representative surveys of U.S. adults, 2001–2002 and 2012–2013. Am J Psychiatry, 178: 548–559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Simms JA, Steensland P, Medina B, Abernathy KE, Chandler LJ, Wise R & Bartlett SE 2008. Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats. Alcohol Clin Exp Res, 32: 1816–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Singletary SJ, Kirsch AJ, Watson J, Karim BO, Huso DL, Hurn PD & Murphy SJ 2005. Lack of correlation of vaginal impedance measurements with hormone levels in the rat. Contemp Top Lab Anim Sci, 44: 37–42. [PMC free article] [PubMed] [Google Scholar]
  38. Tambour S, Brown LL & Crabbe JC 2008. Gender and age at drinking onset affect voluntary alcohol consumption but neither the alcohol deprivation effect nor the response to stress in mice. Alcohol Clin Exp Res, 32: 2100–6. [DOI] [PubMed] [Google Scholar]
  39. Viviani D, Haegler P, Strasser DS & Steiner MA 2012. Sex comparison on long-lasting behavioral and physiological disturbances induced by single shock experience in rats. Physiol Behav, 107: 243–51. [DOI] [PubMed] [Google Scholar]
  40. White AM 2020. Gender differences in the epidemiology of alcohol use and related harms in the United States. Alcohol Res, 40: 01. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wise RA 1973. Voluntary ethanol intake in rats following exposure to ethanol on various schedules. Psychopharmacologia, 29: 203–10. [DOI] [PubMed] [Google Scholar]

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