Cyfip2 allelic variation in C57BL/6J and C57BL/6NJ mice alters free-choice ethanol drinking but not binge-like drinking or wheel-running activity.

BACKGROUND: Since the origin of the C57BL/6 (B6) mouse strain, several phenotypically and genetically distinct B6 substrains have emerged. For example, C57BL/6J mice (B6J) display greater voluntary ethanol consumption and locomotor response to psychostimulants and differences in nucleus accumbens synaptic physiology relative to C57BL/6N (B6N) mice. A non-synonymous serine to phenylalanine point mutation (S968F) in the cytoplasmic FMR1-interacting protein 2 (Cyfip2) gene underlies both the differential locomotor response to cocaine and the accumbal physiology exhibited by these substrains. We examined whether Cyfip2 allelic variation underlies B6 substrain differences in other reward-related phenotypes, such as ethanol intake and wheel-running activity. METHODS: We compared voluntary ethanol consumption, wheel-running, and binge-like ethanol drinking in male and female B6J and B6NJ mice. When substrain differences were observed, additional experiments were performed in two novel mouse models in which the B6N Cyfip2 mutation was either introduced (S968F) into the B6J background or corrected (F968S) via CRISPR/Cas9 technology. RESULTS: B6J consumed significantly more ethanol than B6NJ and allelic variation in Cyfip2 contributed substantially to this substrain difference. In contrast, B6NJ displayed significantly more daily wheel-running than B6J, with Cyfip2 allelic variation playing only a minor role in this substrain difference. Lastly, no substrain differences were observed in binge-like ethanol drinking. CONCLUSIONS: These results contribute to the characterization of behavior-genetic differences between B6 substrains, support previous work indicating that free-choice and binge-like ethanol drinking are dependent on partially distinct genetic networks, and identify a novel phenotypic difference between B6 substrains in wheel-running activity.

Photic Regulation of Circadian Rhythms and Voluntary Ethanol Intake: Role of Melanopsin-expressing Intrinsically Photosensitive Retinal Ganglion Cells.

"Non-image-forming" (NIF) effects of light are mediated primarily by a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment, melanopsin (OPN4). These NIF functions include circadian entrainment, pupillary reflexes, and photic effects on sleep, mood, and cognition. We recently reported that mice of multiple genotypes exhibit reduced voluntary ethanol intake under both constant darkness (DD) and constant light (LL) relative to standard light-dark (LD) conditions. In the present study, we sought to determine whether these effects are mediated by melanopsin-expressing ipRGCs and their potential relationship to photic effects on the circadian system. To this end, we examined the effects of environmental lighting regimen on both ethanol intake and circadian activity rhythms in a genetically engineered mouse model (Opn4aDTA/aDTA) in which melanopsin expression is completely blocked while ipRGCs are progressively ablated due to activation of attenuated diphtheria toxin A (aDTA) transgene under the control of the Opn4 promoter. As expected from previous studies, Opn4aDTA/aDTA mice displayed dramatic attenuation of circadian photosensitivity, but surprisingly, showed identical suppression of ethanol intake under both DD and LL as that seen in controls. These results demonstrate that the effects of lighting regimen on voluntary ethanol intake are independent of melanopsin-expressing ipRGCs and ipRGC-mediated photic effects on the circadian system. Rather, these effects are likely mediated by classical retinal photoreceptors and central pathways.

Suppression of voluntary ethanol intake in mice under constant light and constant darkness.

Seasonal variations in photoperiod are associated with alterations in human mood and behavior. Similarly, manipulation of the environmental lighting regimen can exert pronounced effects on affective behavior in experimental animals. These observations may be due, in part, to light-induced alterations in circadian rhythms, but it seems likely that other, non-circadian factors also contribute. Several studies have shown that voluntary alcohol (ethanol) consumption can be affected by lighting conditions in rodents, suggesting that photoperiodic variation may account for seasonal and geographic patterns of human alcohol consumption. Nevertheless, the existing animal data are somewhat inconsistent, and little work in this area has been performed in mice. In the present study, we monitored circadian activity rhythms and voluntary ethanol consumption under standard 12:12 light-dark (LD) cycles, and in constant light (LL) and constant darkness (DD). Experiment 1 employed male C3H/He inbred mice, while Experiment 2 employed males and females from a genetically heterogeneous line (WSC). Relative to LD conditions, ethanol intake and ethanol preference were reduced under both LL and DD in both experiments. Because similar effects were seen in both LL and DD, neither circadian disruption nor a classical photoperiodic mechanism are likely to account fully for these findings. Instead, we suggest that the absence of circadian entrainment may function as a mild stressor, resulting in reduced ethanol consumption.

Circadian activity rhythms and voluntary ethanol intake in male and female ethanol-preferring rats: effects of long-term ethanol access.

Chronic alcohol (ethanol) intake alters fundamental properties of the circadian clock. While previous studies have reported significant alterations in free-running circadian period during chronic ethanol access, these effects are typically subtle and appear to require high levels of intake. In the present study we examined the effects of long-term voluntary ethanol intake on ethanol consumption and free-running circadian period in male and female, selectively bred ethanol-preferring P and HAD2 rats. In light of previous reports that intermittent access can result in escalated ethanol intake, an initial 2-week water-only baseline was followed by either continuous or intermittent ethanol access (i.e., alternating 15-day epochs of ethanol access and ethanol deprivation) in separate groups of rats. Thus, animals were exposed to either 135 days of continuous ethanol access or to five 15-day access periods alternating with four 15-day periods of ethanol deprivation. Animals were maintained individually in running-wheel cages under continuous darkness throughout the experiment to allow monitoring of free-running activity and drinking rhythms, and 10% (v/v) ethanol and plain water were available continuously via separate drinking tubes during ethanol access. While there were no initial sex differences in ethanol drinking, ethanol preference increased progressively in male P and HAD2 rats under both continuous and intermittent-access conditions, and eventually exceeded that seen in females. Free-running period shortened during the initial ethanol-access epoch in all groups, but the persistence of this effect showed complex dependence on sex, breeding line, and ethanol-access schedule. Finally, while females of both breeding lines displayed higher levels of locomotor activity than males, there was little evidence for modulation of activity level by ethanol access. These results are consistent with previous findings that chronic ethanol intake alters free-running circadian period, and show further that the development of chronobiological tolerance to ethanol may vary by sex and genotype.

Selective breeding for ethanol-related traits alters circadian phenotype.

Previous studies in mice and rats have shown that selective breeding for high and low ethanol preference results in divergence of circadian phenotype in the selected lines. These results indicate that some alleles influencing ethanol preference also contribute to circadian rhythm regulation. Selective breeding has also been used to produce lines of mice differing in a number of other ethanol-related traits, while studies of phenotypic and genetic correlation indicate that diverse ethanol-related traits are influenced by both shared and unshared genetics. In the present study, we examined several features of circadian activity rhythms in a mouse line selected for binge-like drinking and in mouse lines selected for high and low severity of ethanol withdrawal convulsions. Specifically, Experiment 1 compared High Drinking in the Dark (HDID-1) mice to their genetically heterogeneous progenitor line (HS/Npt), and Experiment 2 compared Withdrawal Seizure-Prone (WSP-2) and Withdrawal Seizure-Resistant (WSR-2) mice. Both line pairs displayed differences in their daily activity patterns under light-dark conditions. In addition, HDID-1 mice showed shorter free-running periods in constant light and less coherent activity rhythms across lighting conditions relative to HS/Npt controls, while WSP-2 mice showed longer free-running periods in constant darkness relative to WSR-2 mice. These results strengthen the evidence for genetic linkages between responsiveness to ethanol and circadian regulation, and extend this evidence to include ethanol-related phenotypes other than preference drinking. However, the present results also indicate that the nature of genetic correlations between and within phenotypic domains is highly complex.

Voluntary wheel running attenuates ethanol withdrawal-induced increases in seizure susceptibility in male and female rats.

We recently found that voluntary wheel running attenuated ethanol withdrawal-induced increased susceptibility to chemoconvulsant-induced seizures in male rats. Since female rats recover from ethanol withdrawal (EW) more quickly than male rats across several behavioral measures, this study was designed to determine whether the effects of exercise on EW seizures also exhibited sex differences. Animals were maintained under no-wheel, locked-wheel or free-wheel conditions and ethanol was administered by liquid diet for 14 days with control animals pair-fed an isocaloric diet, after which seizure thresholds were determined at 1 day or 3 days of EW. Consistent with previous reports, females ran significantly more than males, regardless of diet condition. Introduction of the ethanol-containing liquid diet dramatically increased running for females during the day (rest) phase, with little impact on night phase activity. Consistent with previous reports, EW increased seizure susceptibility at 1 day in non-exercising males and females and at 3 days in males. These effects were attenuated by access to running wheels in both sexes. We also assessed the effects of sex, ethanol diet and exercise on ethanol clearance following an acute ethanol administration at 1 day EW in a separate set of animals. Blood ethanol concentrations at 30 min post-injection were lower in males, ethanol-exposed animals, and runners, but no interactions among these factors were detected. Interestingly, females displayed more rapid ethanol clearance than males and there were no effects of either diet or wheel access on clearance rates. Taken together, these data suggest that voluntary wheel running during ethanol administration provides protective effects against EW seizures in both males and females. This effect may be mediated, in part, in male, but not in female rat, by effects of exercise on early pharmacokinetic contributions. This supports the idea that encouraging alcoholics to exercise may benefit their recovery.

Effects of withdrawal from chronic intermittent ethanol vapor on the level and circadian periodicity of running-wheel activity in C57BL/6J and C3H/HeJ mice.

BACKGROUND: Alcohol withdrawal is associated with behavioral and chronobiological disturbances that may persist during protracted abstinence. We previously reported that C57BL/6J (B6) mice show marked but temporary reductions in running-wheel activity, and normal free-running circadian rhythms, following a 4-day chronic intermittent ethanol (CIE) vapor exposure (16 hours of ethanol vapor exposure alternating with 8 hours of withdrawal). In the present experiments, we extend these observations in 2 ways: (i) by examining post-CIE locomotor activity in C3H/HeJ (C3H) mice, an inbred strain characterized by high sensitivity to ethanol withdrawal, and (ii) by directly comparing the responses of B6 and C3H mice to a longer-duration CIE protocol. METHODS: In Experiment 1, C3H mice were exposed to the same 4-day CIE protocol used in our previous study with B6 mice (referred to here as the 1-cycle CIE protocol). In Experiment 2, C3H and B6 mice were exposed to 3 successive 4-day CIE cycles, each separated by 2 days of withdrawal (the 3-cycle CIE protocol). Running-wheel activity was monitored prior to and following CIE, and post-CIE activity was recorded in constant darkness to allow assessment of free-running circadian period and phase. RESULTS: C3H mice displayed pronounced reductions in running-wheel activity that persisted for the duration of the recording period (up to 30 days) following both 1-cycle (Experiment 1) and 3-cycle (Experiment 2) CIE protocols. In contrast, B6 mice showed reductions in locomotor activity that persisted for about 1 week following the 3-cycle CIE protocol, similar to the results of our previous study using a 1-cycle protocol in this strain. Additionally, C3H mice showed significant shortening of free-running period following the 3-cycle, but not the 1-cycle, CIE protocol, while B6 mice showed normal free-running rhythms. CONCLUSIONS: These results reveal genetic differences in the persistence of ethanol withdrawal-induced hypo-locomotion. In addition, chronobiological alterations during extended abstinence may depend on both genetic susceptibility and an extended prior withdrawal history. The present data establish a novel experimental model for long-term behavioral and circadian disruptions associated with ethanol withdrawal.

Running wheel activity protects against increased seizure susceptibility in ethanol withdrawn male rats.

Ethanol withdrawal is a dysphoric condition that arises from termination of ethanol intake by dependent individuals. Common withdrawal symptoms include anxiety, increased reactivity to stimuli and increased seizure susceptibility as well as the risk of increased seizure severity. We use an animal model of dependence and withdrawal to study withdrawal behaviors and potential underlying neurobiological mechanisms. For a number of years, we have quantified pentylenetetrazol seizure thresholds as an assessment of ethanol withdrawal at both one day and three days of withdrawal. Typically, we see a significant decrease in seizure threshold (increased sensitivity to seizure induction) that persists through three days of withdrawal for male rats. Increasing evidence indicates that voluntary exercise affords protection against various challenges to physical and psychological health, including ethanol-related challenges. Therefore, the current study investigated the effect of voluntary wheel running on seizure susceptibility following chronic ethanol administration and withdrawal. We found that voluntary wheel running attenuated the increased sensitivity to pentylenetetrazol-induced seizures observed with ethanol withdrawal, at both the one-day and three-day time points. This result was especially interesting as animals with access to the running wheels consumed more of the ethanol-containing diet. These findings showed that chronic voluntary wheel running reduces the severity of ethanol withdrawal in our animal model and suggest that exercise-based interventions may have some utility in the clinical management of heavy drinking and alcohol withdrawal.