Sex differences in basal motivated behavior, chronic ethanol drinking, and amygdala activity in female and male mice

Abstract

Alcohol use disorder (AUD) is a major public health concern that despite its prevalence, lacks a widely-effective treatment due to the complexity of AUD pathology. AUD is highly comorbid with other psychiatric conditions including anxiety and mood disorders, however it is unclear how these disorders influence each other. The underlying etiology of these comorbidities is difficult to decipher and factors including sex, stress, and the environment further complicate both diagnosis and treatment strategies. To understand more about this bidirectional relationship between AUD and comorbid psychiatric disorders, we ran male and female C57Bl/6j mice through baseline behavioral testing followed by intermittent access-two bottle choice (IA-2BC) drinking. We found no sex differences in basal anxiety-like or depressive-like behavior, however females displayed enhanced motivated feeding behavior. Females consumed more ethanol than males, at both 1hr and 24hr timepoints. Basal affective state did not predict subsequent ethanol intake in either sex, however exploratory behavior was positively correlated with drinking in males but not females. We then re-assessed negative affect behavior following chronic ethanol drinking to determine if drinking impacted subsequent affective behavior and found no relationship between ethanol intake and affective state in males or females. We also examined how chronic ethanol drinking affected central amygdala (CeA) and basolateral amygdala (BLA) neuronal activity in males and females. Ethanol-drinking females had a decrease in CeA neuronal activity, driven by reduced activity in the lateral (CeA_l) sub-region, while in males there was no significant difference in CeA activity compared to water controls. Neither males or females had a significant change in BLA neuronal activity following chronic ethanol drinking. Collectively, these results demonstrate sex differences in basal motivated behavior, drinking behavior, and subregion-specific amygdala neuronal activity following chronic ethanol drinking which may inform the sex differences seen in patients diagnosed with AUD and comorbid conditions.

Keywords: Alcohol; Anxiety; BLA; CeA; Female; Male.

Fig. 1

A. Baseline behavioral testing timeline highlighting when each test was conducted (indicated by days) relative to the start of the intermittent access – two bottle choice (IA-2BC) drinking paradigm. B. Diagram of the splash test. C. Basal latency to groom time measured in males and females, n=16M, 15F. Unpaired t-test: p=0.5266. D. Basal total time spent grooming measured in males and females, n=16M, 15F. Unpaired t-test: p=0.2020. E. Basal total time spent rearing measured in males and females, n=16M, 15F. Unpaired t-test: p=0.6554. F. Diagram of the novelty-suppressed feeding (NSF) test. G. Basal latency to feed time measured in males and females, n=16M, 15F. Unpaired t-test: p=0.7553. H. Diagram of the NSF post-test. I. Basal post-test food consumption measured in males and females, n=16M, 15F. Unpaired t-test: *p=0.0034.

Fig. 2

A. Diagram of the intermittent access – two bottle choice (IA-2BC) drinking paradigm. B. Complete experimental timeline, highlighting the weeks when mice were drinking ethanol. C. 24hr ethanol intake in males and females during the IA-2BC paradigm. n=9M, 7F. 2-Way ANOVA: significant interaction (*#p=0.0038, F (21, 292)=2.1), main effect of sex (*p=0.0001, F (1, 14)=27.24) and time (#p<0.0001, F (6.6, 91.71)=10.13). D. 1hr ethanol intake in males and females. n=9M, 7F. 2-Way ANOVA: main effect of sex (*p=0.0004, F (1, 14)=21.10) and time (#p=0.0094, F (3.94, 55.15)=3.75). E. 24hr total water intake in males and females. n=9M, 7F. 2-Way ANOVA: main effect of time (#p=0.0012, F (5.63, 78.26)=4.25). F. 24hr ethanol preference in males and females. n=9M, 7F. 2-Way ANOVA: main effect of time (#p=0.0005, F (5.92, 81.79)=4.55). G. Association between latency to groom time and week 1 average ethanol intake in males and females. n=9M, 7F. Simple linear regression: (Male: R²=0.0181, p=0.7303, Female: R²=0.0184, p=0.7717). H. Association between time spent rearing and week 1 average ethanol intake in males and females. n=9M, 7F. Simple linear regression: (Male: R²=0.4635, *p=0.0435, Female: R²=0.1159, p=0.4549). I. Association between latency to feed time and week 1 average ethanol intake in males and females. n=9M, 7F. Simple linear regression: (Male: R²=0.0314, p=0.6482, Female: R²=0.1755, p=0.3495). J. Association between latency to groom time and weeks 1–8 average ethanol intake in males and females. n=9M, 7F. Simple linear regression: (Male: R²=0.2958, p=0.1301, Female: R²=0.0119, p=0.8162). K. Association between time spent rearing and weeks 1–8 average ethanol intake in males and females. n=9M, 7F. Simple linear regression: (Male: R²=0.5234, *p=0.0276, Female: R²=0.0025, p=0.9157). L. Association between latency to feed time and weeks 1–8 average ethanol intake in males and females. n=9M, 7F. Simple linear regression: (Male: R²=0.1544, p=0.2955, Female: R²=0.1335, p=0.4203).

Fig. 3

A. Diagram of the splash test. B. Behavioral testing timeline highlighting when the splash test was conducted relative to when bottles were taken off at the end of IA-2BC week 5 and put on at the start of IA-2BC week 6. C. Latency to groom time measured in female and male water and ethanol groups after 5 weeks of voluntary drinking. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. 2-Way ANOVA: main effect of treatment (*p=0.0186, F (1, 27)=6.274), in females: *p=0.0119. D. Time spent grooming measured in female and male water and ethanol groups after 5 weeks of voluntary drinking. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. 2-Way ANOVA: main effect of sex (*p=0.0003, F (1, 27)=17.46), water: *p=0.0073, ethanol: *p=0.0056. E. Time spent rearing measured in female and male water and ethanol groups after 5 weeks of voluntary drinking. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. 2-Way ANOVA: (p=0.6694, F (1, 27)=1.1864). F. Latency to groom time measured in female water and ethanol groups before and after 5 weeks of voluntary drinking. n=8 water, 7 ethanol. 2-Way ANOVA: main effect of time (*p=0.0171, F (1, 13)=7.47). G. Time spent grooming measured in female water and ethanol groups before and after 5 weeks of voluntary drinking. n=8 water, 7 ethanol. 2-Way ANOVA: main effect of time (*p=0.0023, F (1, 13)=14.35). H. Time spent rearing measured in female water and ethanol groups before and after 5 weeks of voluntary drinking. n=8 water, 7 ethanol. 2-Way ANOVA: main effect of time (*p<0.0001, F (1, 13)=35.90). I. Latency to groom time measured in male water and ethanol groups before and after 5 weeks of voluntary drinking. n=7 water, 9 ethanol. 2-Way ANOVA: main effect of time (*p=0.0019, F (1, 14)=14.59). J. Time spent grooming measured in male water and ethanol groups before and after 5 weeks of voluntary drinking. n=7 water, 9 ethanol. 2-Way ANOVA: p=0.1730. K. Time spent rearing measured in male water and ethanol groups before and after 5 weeks of voluntary drinking. n=7 water, 9 ethanol. 2-Way ANOVA: main effect of time (*p=0.0213, F (1, 14)=6.714). L. Association between weeks 1–5 average ethanol intake and latency to groom time in females and males. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. Simple linear regression: (Female: R²=0.1813, p=0.3408, Male: R²=0.0454, p=0.5822). M. Association between weeks 1–5 average ethanol intake and time spent grooming in females and males. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. Simple linear regression: (Female: R²=0.0085, p=0.8444, Male: R²=0.1199, p=0.3613). N. Association between weeks 1–5 average ethanol intake and time spent rearing in females and males. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. Simple linear regression: (Female: R²=0.0103, p=0.8283, Male: R²=0.0299, p=0.6566).

Fig. 4

A. Behavioral testing timeline highlighting when the novelty-suppressed feeding (NSF) test was conducted relative to when bottles were taken off at the end of IA-2BC week 6 and put on at the start of IA-2BC week 7. B. Diagram of the NSF test. C. Diagram of the NSF post-test. D. Latency to feed time measured in female water and ethanol groups before and after 6 weeks of voluntary drinking. n=8 water, 7 ethanol. 2-Way ANOVA: p=0.1732. E. Post-test food consumption measured in female water and ethanol groups before and after 6 weeks of voluntary drinking. n=8 water, 7 ethanol. 2-Way ANOVA: main effect of time (*p<0.0001, F (1, 26)=43.19). F. Latency to feed time measured in male water and ethanol groups before and after 6 weeks of voluntary drinking. n=8 water, 7 ethanol. 2-Way ANOVA: interaction: p=0.4011. G. Post-test food consumption measured in male water and ethanol groups before and after 6 weeks of voluntary drinking. n=7 water, 9 ethanol. 2-Way ANOVA: main effect of time (*p=0.0001, F (1, 28)=20.02). H. Association between weeks 1–6 average ethanol intake and latency to feed time in females and males. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. Simple linear regression: (Female: R²=0.2098, p=0.3013, Male: R²=0.1700, p=0.2702). I. Association between weeks 1–6 average ethanol intake and post-test food consumption in females and males. Female: n=8 water, 7 ethanol. Male: n=7 water, 9 ethanol. Simple linear regression: (Female: R²=0.2777, p=0.2242, Male: R²=0.3430, p=0.0975).

Fig. 5

A. Representative image showing cFos expression (red) in the central amygdala (CeA) with the lateral (CeA_l) and medial (CeA_m) regions outlined. Scale bar=50μm. B. Representative image showing CeA cFos expression (red) and DAPI staining (blue) in females (top) and males (bottom) that were exposed to chronic ethanol or water. Scale bars=50μm. C. CeA cFos expression averaged by animal in female and male water and ethanol groups. Female: n=8 water, 7 ethanol. Male: n=6 water, 9 ethanol. 2-Way ANOVA: main effect of treatment (*p=0.0182, F (1, 26)=6.347), females (*p=0.0336). D. CeA_m cFos expression averaged by animal in female and male water and ethanol groups. Female: n=8 water, 7 ethanol. Male: n=6 water, 9 ethanol. 2-Way ANOVA: main effect of treatment (*p=0.0183, F (1, 26)=6.337). E. CeA_l cFos expression averaged by animal in female and male water and ethanol groups. Female: n=8 water, 7 ethanol. Male: n=6 water, 9 ethanol. 2-Way ANOVA: main effect of treatment (*p=0.0062, F (1, 26)=8.886), females (*p=0.0149). F. Association between weeks 1–8 average ethanol intake and CeA cFos+ cells in females and males. Female: n=7, Male: n=9. Simple linear regression: (Female: R²=0.0891, p=0.5156, Male: R²=0.1502, p=0.3027). G. Association between weeks 1–8 average ethanol intake and CeA_m cFos+ cells in females and males. Female: n=7, Male: n=9. Simple linear regression: (Female: R²=0.1574, p=0.3782, Male: R²=0.1347, p=0.3313). H. Association between weeks 1–8 average ethanol intake and CeA_m cFos+ cells in females and males. Female: n=7, Male: n=9. Simple linear regression: (Female: R²=0.0568, p=0.6067, Male: R²=0.1240, p=0.3527).

Fig. 6

A. Representative image showing cFos expression (red) in the basolateral amygdala (BLA). Scale bar=50μm. B. Representative image showing BLA cFos expression (red) and DAPI staining (blue) in females (top) and males (bottom) that were exposed to chronic ethanol or water. Scale bars=50μm. C. BLA cFos expression averaged by animal in female and male water and ethanol groups. Female: n=8 water, 7 ethanol. Male: n=6 water, 9 ethanol. 2-Way ANOVA: p=0.7745. D. Association between weeks 1–8 average ethanol intake and BLA cFos+ cells in females and males. Female: n=7, Male: n=9. Simple linear regression: (Female: R²=0.2543, p=0.2484, Male: R²=0.4254, p=0.0569).