Affective Disruption During Forced Ethanol Abstinence in C57BL/6J and C57BL/6NJ Mice.

BACKGROUND: In alcohol-dependent individuals, acute alcohol withdrawal results in severe physiological disruption, including potentially lethal central nervous system hyperexcitability. Although benzodiazepines successfully mitigate such symptoms, this treatment does not significantly reduce recidivism rates in postdependent individuals. Instead, persistent affective disturbances that often emerge weeks to months after initial detoxification appear to play a significant role in relapse risk; however, it remains unclear whether genetic predispositions contribute to their emergence, severity, and/or duration. Interestingly, significant genotypic and phenotypic differences have been observed among distinct C57BL/6 (B6) substrains, and, in particular, C57BL/6J (B6J) mice have been found to reliably exhibit higher voluntary ethanol (EtOH) intake and EtOH preference compared to several C57BL/6N (B6N)-derived substrains. To date, however, B6 substrains have not been directly compared on measures of acute withdrawal severity or affective-behavioral disruption during extended abstinence. METHODS: Male and female B6J and B6NJ mice were exposed to either a 7-day chronic intermittent EtOH vapor (CIE) protocol or to ordinary room air in inhalation chambers. Subsequently, blood EtOH concentrations and handling-induced convulsions were evaluated during acute withdrawal, and mice were then tested weekly for affective behavior on the sucrose preference test, light-dark box test, and forced swim test throughout 4 weeks of (forced) abstinence. RESULTS: Despite documented differences in voluntary EtOH intake between these substrains, we found little evidence for substrain differences in either acute withdrawal or long-term abstinence between B6J and B6NJ mice. CONCLUSIONS: In B6J and B6NJ mice, both the acute and long-term sequelae of EtOH withdrawal are dependent on largely nonoverlapping gene networks relative to those underlying voluntary EtOH drinking.

Affective Behavior in Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant Mice during Long-Term Alcohol Abstinence.

BACKGROUND: While the acute alcohol withdrawal syndrome has been well characterized both in human clinical studies and in experimental animals, much less is known regarding long-term affective disturbances that can sometimes persist during protracted abstinence. Nevertheless, since relapse often occurs long after acute detoxification and may be predicted by persistent affective disruption, a better understanding of the long-term behavioral consequences of prior alcohol dependence may lead to improved strategies for relapse prevention. METHODS: Male and female Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice from the second selection replicate (WSP-2, WSR-2) were exposed to a 10-day chronic-intermittent ethanol vapor protocol (CIE) or plain air and then tested repeatedly on the sucrose preference test (SPT), marble burying test (MBT), and the light-dark box test (LDT) over 7 weeks of (forced) abstinence. RESULTS: While WSP and WSR mice differed significantly on tests of anxiety-like behavior (LDT, MBT), we found little evidence for long-term affective disruption following CIE in either line. The major exception was in the LDT, in that WSP but not WSR mice displayed longer latencies to enter the light compartment following CIE relative to air-controls. CONCLUSIONS: Selective breeding for acute withdrawal severity has resulted in differences in anxiety-like behavior between WSP and WSR mice. In contrast, however, genes contributing to the severity of acute withdrawal convulsions appear to have little overlap with those predisposing to affective disruption during long-term abstinence.

Chronic shift-lag alters the circadian clock of NK cells and promotes lung cancer growth in rats.

Prolonged subjection to unstable work or lighting schedules, particularly in rotating shift-workers, is associated with an increased risk of immune-related diseases, including several cancers. Consequences of chronic circadian disruption may also extend to the innate immune system to promote cancer growth, as NK cell function is modulated by circadian mechanisms and plays a key role in lysis of tumor cells. To determine if NK cell function is disrupted by a model of human shift-work and jet-lag, Fischer (344) rats were exposed to either a standard 12:12 light-dark cycle or a chronic shift-lag paradigm consisting of 10 repeated 6-h photic advances occurring every 2 d, followed by 5-7 d of constant darkness. This model resulted in considerable circadian disruption, as assessed by circadian running-wheel activity. NK cells were enriched from control and shifted animals, and gene, protein, and cytolytic activity assays were performed. Chronic shift-lag altered the circadian expression of clock genes, Per2 and Bmal1, and cytolytic factors, perforin and granzyme B, as well as the cytokine, IFN-γ. These alterations were correlated with suppressed circadian expression of NK cytolytic activity. Further, chronic shift-lag attenuated NK cell cytolytic activity under stimulated in vivo conditions, and promoted lung tumor growth following i.v. injection of MADB106 tumor cells. Together, these findings suggest chronic circadian disruption promotes tumor growth by altering the circadian rhythms of NK cell function.

Escalation of intake under intermittent ethanol access in diverse mouse genotypes.

Experimental animals offered continuous 24-hour free choice access to ethanol rarely display voluntary ethanol consumption at levels sufficient to induce intoxication or to engender dependence. One of the simplest ways to increase voluntary ethanol intake is to impose temporal limitations on ethanol availability. Escalation of ethanol intake has been observed in both rats and mice under a variety of different schedules of alternating ethanol access and deprivation. Although such effects have been observed in a variety of rat and mouse genotypes, little is known concerning possible genetic correlations between responses to intermittent ethanol access and other ethanol-related phenotypes. In the present study, we examined the effects of intermittent ethanol access in mouse genotypes characterized by divergent responses to ethanol in other domains, including ethanol preference (C57BL/6J and C3H/HeJ mice), binge-like ethanol drinking (High Drinking in the Dark and HS/Npt mice) and ethanol withdrawal severity (Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice). Although intermittent ethanol access resulted in escalated ethanol intake in all tested genotypes, the robustness of the effect varied across genotypes. On the other hand, we saw no evidence that the effects of intermittent access are correlated with either binge-like drinking or withdrawal severity, and only weak evidence for a genetic correlation with baseline ethanol preference. Thus, these different ethanol-related traits appear to depend on largely unique sets of genetic mediators.

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.

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.

Neurobiology of Circadian Rhythm Regulation.

In this review, we provide a summary of the field of mammalian circadian neurobiology circa 2015. While many additional details have emerged in the intervening 7 years, understanding of the fundamental structure and function of this critical neural system remains intact. Thus, the present review continues to provide a valuable introduction for those seeking an integrative multilevel overview of the circadian system. In brief, the circadian system comprises a coupled network of molecular/cellular- and tissue-level oscillators, hierarchically coordinated by the hypothalamic suprachiasmatic nuclear circadian pacemaker, and entrained by both photic and nonphotic signals.

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.

Chronic ethanol intake alters circadian phase shifting and free-running period in mice.

Chronic alcohol intake is associated with widespread disruptions in sleep and circadian rhythms in both human alcoholics and in experimental animals. Recent studies have demonstrated that chronic and acute ethanol treatments alter fundamental properties of the circadian pacemaker--including free-running period and responsiveness to photic and nonphotic phase-shifting stimuli--in rats and hamsters. In the present work, the authors extend these observations to the C57BL/6J mouse, an inbred strain characterized by very high levels of voluntary ethanol intake and by reliable and stable free-running circadian activity rhythms. Mice were housed individually in running-wheel cages under conditions of either voluntary or forced ethanol intake, whereas controls were maintained on plain water. Forced ethanol intake significantly attenuated photic phase delays (but not phase advances) and shortened free-running period in constant darkness, but voluntary ethanol intake failed to affect either of these parameters. Thus, high levels of chronic ethanol intake, beyond those normally achieved under voluntary drinking conditions, are required to alter fundamental circadian pacemaker properties in C57BL/6J mice. These observations may be related to the relative ethanol insensitivity displayed by this strain in several other phenotypic domains, including ethanol-induced sedation, ataxia, and withdrawal. Additional experiments will investigate chronobiological sensitivity to ethanol in a range of inbred strains showing diverse ethanol-related phenotypes.

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.

Chronic ethanol intake modulates photic and non-photic circadian phase responses in the Syrian hamster.

Chronic alcohol intake disrupts sleep and other circadian biological rhythms in both human alcoholics and in experimental animals. Recent studies from our laboratory indicate that these effects may be due, in part, to ethanol-induced alterations in fundamental properties of the circadian pacemaker. The present study explored the effects of chronic voluntary ethanol intake (25% v/v) on circadian phase responses to both photic and non-photic stimuli in Syrian hamsters. Hamsters were used in these experiments because they are a popular model organism in behavioral chronobiology research, and are characterized by unusually high levels of voluntary ethanol intake. Relative to controls, ethanol-exposed animals showed attenuation of circadian phase responses and wheel running activity following acute administration of the benzodiazepine, triazolam, a non-photic phase-shifting stimulus. In addition, ethanol-exposed animals displayed reduced phase advances, but normal phase delays, in response to brief light pulses. While the mechanisms underlying these effects remain to be elucidated, we hypothesize that ionotropic GABA and glutamate receptors may be involved, since these proteins serve as important targets for the neurobiological effects of ethanol, and are also known to be critically involved in the modulation of photic and non-photic circadian phase responses.

Effects of ethanol intake and ethanol withdrawal on free-running circadian activity rhythms in rats.

Chronic alcohol intake and alcohol withdrawal are associated with dramatic disruptions of daily (circadian) biological rhythms in both human alcoholics and experimental animals. The extent to which these observations are due to pharmacological effects on the underlying circadian pacemaker is not known, however, since no human studies and very few animal studies have been conducted under free-running conditions. In the present study, free-running circadian activity (wheel-running) rhythms of rats were monitored before, during and after exposure to either 10% or 20% ethanol solution as the only drinking fluid. Across individuals, both lengthening and shortening of free-running period were observed during ethanol intake, and treatment termination led to either a return to baseline or to an exacerbation of the original ethanol effect. These variable effects appeared to be related to both ethanol concentration and to individual differences in baseline period, such that relatively short free-running period during baseline was associated with greater period-lengthening during ethanol exposure. These bidirectional affects of ethanol on free-running period are generally similar to effects seen previously with other psychoactive drugs, including antidepressants. The results of this study indicate that ethanol influences the circadian pacemaker, and that the chronobiological disruptions seen in human alcoholics may be due, in part, to alterations in circadian pacemaker regulation.

Chronic ethanol intake alters circadian period-responses to brief light pulses in rats.

Although chronic alcohol intake is associated with widespread disruptions of sleep-wake cycles and other daily biological rhythms in both human alcoholics and experimental animals, the extent to which the chronobiological effects of alcohol are mediated by effects on the underlying circadian pacemaker remains unknown. Nevertheless, recent studies indicate that both adult and perinatal ethanol treatments may alter the free-running period and photic responsiveness of the circadian pacemaker. The present experiment was designed to further characterize the effects of chronic ethanol intake on the response of the rat circadian pacemaker to brief light pulses. Ethanol-treated and control animals were exposed to 15-min light pulses during either early or late subjective night on the first day of constant darkness following entrainment to a 12:12 light-dark cycle. Relative to pulses delivered during early subjective night and to "no-pulse" conditions, light pulses delivered during late subjective night resulted in period-shortening after-effects under constant darkness, but only in control animals, not in ethanol-treated animals. These results indicate that chronic ethanol intake reduces the responsiveness of the circadian pacemaker to acute photic stimulation, and suggest that the chronobiological disruptions seen in human alcoholics are due in part to alterations in circadian pacemaker function.

Circadian activity rhythms in selectively bred ethanol-preferring and nonpreferring rats.

Chronic alcohol intake is associated with dramatic disruptions in sleep and other circadian biological rhythms in both humans and experimental animals. In human alcoholics, these disruptions persist during extended abstinence and appear to promote relapse to drinking. Whereas chronic ethanol intake alters fundamental properties of the circadian pacemaker in unselected rats, nothing is known concerning circadian pacemaker function in selectively bred ethanol-preferring and nonpreferring rats, which are the most widely accepted animal models of genetic predisposition to alcoholism. The present experiments were designed to characterize free-running circadian activity (wheel-running) rhythms under both constant darkness and constant light in selectively bred ethanol-preferring (P, HAD2) and nonpreferring (NP, LAD2) rats. Differences in circadian organization between ethanol-preferring and nonpreferring animals were seen for both pairs of selected lines (P vs. NP; HAD2 vs. LAD2), but these differences were not identical in the two line pairs. For example, although P rats showed shorter free-running periods than NP rats only in constant light, HAD2 rats showed shorter free-running periods than LAD2 rats only in constant darkness. In addition, ethanol-preferring HAD2 rats showed a high rate of rhythm "splitting" that was not seen in any of the other three lines. Taken together, these results suggest that the circadian pacemakers of P and NP rats differ mainly in light sensitivity, whereas those of HAD2 and LAD2 rats differ in their intrinsic period.

Phase shifting the hamster circadian clock by 15-minute dark pulses.

The mammalian circadian pacemaker can be phase shifted by exposure to a period of darkness interrupting otherwise continuous light. Circadian phase shifting by dark pulses was interpreted originally as reflecting a photic mirror-image mechanism, but more recent observations suggest that dark pulse-induced phase shifting may be mediated by a nonphotic, behavioral state-dependent mechanism. The authors recently presented evidence indicating that the dark-pulse phase response curve (PRC) is in fact a complex function, reflecting both photic mirror image and nonphotic mechanisms at different phases of the circadian cycle. Previous studies of dark pulse-induced phase shifting have universally employed relatively long (2 to 6 h) pulses, which complicates PRC analysis due to the extended segment of the underlying PRC spanned by such a long pulse. The present study was therefore designed to examine the phase-shifting effects of brief 15-min dark pulses presented at both mid-subjective day and subjective dusk, and to explore the possible activity dependence of these effects by using physical restraint to prevent evoked locomotor activity. The results indicate that 15-min dark pulses are effective phase-shifting stimuli at both midday and dusk. Furthermore, as with longer dark pulses, phase shifting by 15-min dark pulses is completely blocked by physical restraint during subjective day but combines in a simple additive manner with the independent phase-shifting effect of restraint at subjective dusk.

Chronobiology of ethanol: animal models.

Clinical and epidemiological observations have revealed that alcohol abuse and alcoholism are associated with widespread disruptions in sleep and other circadian biological rhythms. As with other psychiatric disorders, animal models have been very useful in efforts to better understand the cause and effect relationships underlying the largely correlative human data. This review summarizes the experimental findings indicating bidirectional interactions between alcohol (ethanol) consumption and the circadian timing system, emphasizing behavioral studies conducted in the author's laboratory. Together with convergent evidence from multiple laboratories, the work summarized here establishes that ethanol intake (or administration) alters fundamental properties of the underlying circadian pacemaker. In turn, circadian disruption induced by either environmental or genetic manipulations can alter voluntary ethanol intake. These reciprocal interactions may create a vicious cycle that contributes to the downward spiral of alcohol and drug addiction. In the future, such studies may lead to the development of chronobiologically based interventions to prevent relapse and effectively mitigate some of the societal burden associated with such disorders.

Neurobiology of Circadian Rhythm Regulation.

Over the past few decades, multilevel research has elucidated the basic neuroanatomy, neurochemistry, and molecular neurobiology of the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). The circadian timing system is composed of a large number of cellular oscillators located in the SCN, in non-SCN brain structures, and throughout the body. Cellular-level oscillations are generated by a molecular feedback loop in which circadian clock genes rhythmically regulate their own transcription, as well as that of hundreds of clock-controlled genes. The maintenance of proper coordination within this network of cellular- and tissue-level clocks is essential for health and well-being.

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.

Chronobiology of alcohol: studies in C57BL/6J and DBA/2J inbred mice.

Human alcoholics display dramatic disruptions of circadian rhythms that may contribute to the maintenance of excessive drinking, thus creating a vicious cycle. While clinical studies cannot establish direct causal mechanisms, recent animal experiments have revealed bidirectional interactions between circadian rhythms and ethanol intake, suggesting that the chronobiological disruptions seen in human alcoholics are mediated in part by alterations in circadian pacemaker function. The present study was designed to further explore these interactions using C57BL/6J (B6) and DBA/2J (D2) inbred mice, two widely employed strains differing in both circadian and alcohol-related phenotypes. Mice were maintained in running-wheel cages with or without free-choice access to ethanol and exposed to a variety of lighting regimens, including standard light-dark cycles, constant darkness, constant light, and a "shift-lag" schedule consisting of repeated light-dark phase shifts. Relative to the standard light-dark cycle, B6 mice showed reduced ethanol intake in both constant darkness and constant light, while D2 mice showed reduced ethanol intake only in constant darkness. In contrast, shift-lag lighting failed to affect ethanol intake in either strain. Access to ethanol altered daily activity patterns in both B6 and D2 mice, and increased activity levels in D2 mice, but had no effects on other circadian parameters. Thus, the overall pattern of results was broadly similar in both strains, and consistent with previous observations that chronic ethanol intake alters circadian activity patterns while environmental perturbation of circadian rhythms modulates voluntary ethanol intake. These results suggest that circadian-based interventions may prove useful in the management of alcohol use disorders.

Sex and regional differences in effects of chronic intermittent ethanol exposure on subsequent excitotoxic challenges in hippocampal slice cultures.

The organotypic hippocampal slice culture technique was used to study how the effects of repeated ethanol withdrawal might differ between males and females at the cellular level, including potential modulation of subsequent insults. A chronic intermittent ethanol (CIE) exposure paradigm was employed, with 3 days of exposure followed by 24 h withdrawal for 3 cycles. Slices were next exposed to corticosterone (CORT) or pentylenetetrazol (PTZ) for 24 h then imaged for propidium iodide (PI) signal intensities. There were sex-selective responses in the CA1 region and dentate gyrus of the hippocampal slice cultures to treatment with CIE and/or CORT or PTZ. The 50 mM CIE alone generally did not increase the PI signal, but enhanced sensitivity to the toxic effects of CORT (particularly for females) and PTZ (particularly for males). In contrast, 100 mM CIE elicited a toxic response that was greater in females than males, and was exacerbated by exposure to PTZ. These data showed that hippocampal sexual dimorphism influences sensitivity to ethanol and other toxic chemicals even in an immature state. Low-dose CIE may attenuate harm from additional challenges in a hippocampal sex- and region-selective manner. These findings add to the growing evidence of important neurobiological sex differences in responses to chronic ethanol exposure and withdrawal.