DID it or DIDn't it? Exploration of a failure to replicate binge-like alcohol-drinking in C57BL/6J mice

Abstract

We previously reported that commercially-sourced C57BL/6J (B6) male mice with a history of adult-onset binge-drinking exhibit anxiety-like behavior in early withdrawal, while the negative affective state incubates during protracted withdrawal in adolescent-onset binge-drinking males. As the results of such studies are potentially confounded by age-related differences in reactivity to environmental stress, we employed a 2-bottle-choice DID procedure (20 and 40% alcohol; 20 min habituation to the drinking cage) to examine the effects of binge-drinking on negative affect in male and female, adult and adolescent, B6 mice from our university colony. Unexpectedly, the mice in the initial experiment exhibited very low alcohol intake, with little sign of withdrawalinduced negative affect. This failure to replicate prompted us to examine how the duration of drinking cage habituation, the number of alcohol concentrations presented and the animal source might influence the propensity to binge-drink. Herein, we show that both male and female adult mice from our colony will binge-drink when allowed 45 min to habituate to the drinking cages, irrespective of whether mice are offered a choice between 2, 3 or 4 alcohol concentrations. Further, when drinking under 4-bottle-choice procedures (5, 10, 20 and 40% alcohol), adult-onset binge-drinking females exhibit robust negative affect in early withdrawal akin to that reported previously for adult males; however, the negative affective state persists for at least 30 days into withdrawal. Also unlike males, adolescent-onset binge-drinking females exhibit some signs of negative affect, as well as potentiated alcohol intake, in early withdrawal, which persist into later withdrawal. These latter data suggest that the age-related differences in the temporal patterning of the negative affective state produced by alcohol withdrawal may vary as a function of sex, which may have implications for understanding sex differences in the etiology of affective disorders and alcoholism co-morbidity.

Chronic Voluntary Binge Ethanol Consumption Causes Sex‐Specific Differences in Microglial Signaling Pathways and Withdrawal‐associated Behaviors in Mice

“…Similarly, male rats following 14 days of EtOH consumption exhibited greater startle responses than female or ovariectomized female rats (Reilly et al, 2009). On the other hand, although only male mice were tested and found to exhibit withdrawal-related anxiety behaviors in the original study (Lee et al, 2015), a very recent follow-up study showed that refinements of their DID paradigm resulted in the exhibition of withdrawal-related anxiety behaviors by both male and female mice, albeit some differences in persistence of the withdrawal between the sexes (Szumlinski et al, 2019). These observations highlighted the challenges in the measurement of subtle behavioral abnormalities such as those related to the DID EtOH withdrawal, the differences in various animal model and behavioral test designs, and the contribution of cohort size of test animals.…”

Incubation of Negative Affect during Protracted Alcohol Withdrawal Is Age-, but Not Sex-Selective

“…Female laboratory rodents also escalate alcohol-taking more quickly and exhibit greater relapse-like drinking than their male counterparts [ 30 , 31 , 32 ]. While fewer in number, some animal studies have examined the interactions between biological sex and age of drinking-onset with respect to binge-drinking-related outcomes [ 32 , 33 , 34 , 35 , 36 ]. Of direct relevance to this project, we demonstrated recently that akin to adult males [ 10 ], adult female B6 mice with a 2-week history of binge-drinking exhibit robust sings of negative affect during early (1 day) withdrawal [ 35 ].…”

“…While fewer in number, some animal studies have examined the interactions between biological sex and age of drinking-onset with respect to binge-drinking-related outcomes [ 32 , 33 , 34 , 35 , 36 ]. Of direct relevance to this project, we demonstrated recently that akin to adult males [ 10 ], adult female B6 mice with a 2-week history of binge-drinking exhibit robust sings of negative affect during early (1 day) withdrawal [ 35 ]. However, in contrast to males [ 11 , 25 ], this negative affective state persists in adult females for at least 30 days post-drinking [ 35 ].…”

“…Of direct relevance to this project, we demonstrated recently that akin to adult males [ 10 ], adult female B6 mice with a 2-week history of binge-drinking exhibit robust sings of negative affect during early (1 day) withdrawal [ 35 ]. However, in contrast to males [ 11 , 25 ], this negative affective state persists in adult females for at least 30 days post-drinking [ 35 ]. We also detected what might be a sex difference in the onset of a negative affective state during withdrawal in binge-drinking adolescents; female mice with a prior history of adolescent binge-drinking exhibit signs of a negative affective state in both early and later withdrawal [ 35 ], while that observed in males incubates with the passage of time post-drinking [ 24 ].…”

“…However, in contrast to males [ 11 , 25 ], this negative affective state persists in adult females for at least 30 days post-drinking [ 35 ]. We also detected what might be a sex difference in the onset of a negative affective state during withdrawal in binge-drinking adolescents; female mice with a prior history of adolescent binge-drinking exhibit signs of a negative affective state in both early and later withdrawal [ 35 ], while that observed in males incubates with the passage of time post-drinking [ 24 ]. Taken together, these findings regarding the interaction between sex and age of binge-drinking onset suggest that females may be more susceptible than males to developing a long-lasting change in emotionality during protracted alcohol withdrawal that warranted a direct investigation.…”