Sensitivity and Resilience to Predator Stress-Enhanced Ethanol Drinking Is Associated With Sex-Dependent Differences in Stress-Regulating Systems.

Stress can increase ethanol drinking, and evidence confirms an association between post-traumatic stress disorder (PTSD) and the development of alcohol use disorder (AUD). Exposure to predator odor is considered a traumatic stressor, and predator stress (PS) has been used extensively as an animal model of PTSD. Our prior work determined that repeated exposure to intermittent PS significantly increased anxiety-related behavior, corticosterone levels, and neuronal activation in the hippocampus and prefrontal cortex in naïve male and female C57BL/6J mice. Intermittent PS exposure also increased subsequent ethanol drinking in a subgroup of animals, with heterogeneity of responses as seen with comorbid PTSD and AUD. The present studies built upon this prior work and began to characterize "sensitivity" and "resilience" to PS-enhanced drinking. Ethanol drinking was measured during baseline, intermittent PS exposure, and post-stress; mice were euthanized after 24-h abstinence. Calculation of median and interquartile ranges identified "sensitive" (>20% increase in drinking over baseline) and "resilient" (no change or decrease in drinking from baseline) subgroups. Intermittent PS significantly increased subsequent ethanol intake in 24% of male (↑60%) and in 20% of female (↑71%) C57BL/6J mice in the "sensitive" subgroup. Plasma corticosterone levels were increased significantly after PS in both sexes, but levels were lower in the "sensitive" vs. "resilient" subgroups. In representative mice from "sensitive" and "resilient" subgroups, prefrontal cortex and hippocampus were analyzed by Western Blotting for levels of corticotropin releasing factor (CRF) receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor, vs. separate naïve age-matched mice. In prefrontal cortex, CRF receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor levels were significantly higher in "sensitive" vs. naïve and "resilient" mice only in females. In hippocampus, CRF receptor 1, CRF receptor 2 and glucocorticoid receptor levels were significantly lower in "resilient" vs. naïve and "sensitive" mice across both sexes. These results indicate that sex strongly influences the effects of ethanol drinking and stress on proteins regulating stress and anxiety responses. They further suggest that targeting the CRF system and glucocorticoid receptors in AUD needs to consider the comorbidity of PTSD with AUD and sex of treated individuals.

Sexually divergent changes in select brain proteins and neurosteroid levels after a history of ethanol drinking and intermittent PTSD-like stress exposure in adult C57BL/6J mice.

Human studies reported that the number of past-year stressors was positively related to current drinking patterns, including binge drinking. In animal models, exposure to predator odor stress (PS), considered a model of traumatic stress, consistently increased ethanol intake. Recently, we reported that repeated PS significantly increased ethanol intake and had a synergistic interaction with prior binge drinking (binge group) in male but not in female C57BL/6J mice, when compared to mice without prior binge exposure (control group). The current studies utilized plasma and dissected prefrontal cortex (PFC) and hippocampal tissue from these animals and from age-matched naïve mice (naïve group). Western blots assessed relative protein levels of P450scc (an enzyme involved in the first step of steroidogenesis), of GABAA receptor α2 and α4 subunits, and of two proteins involved in synaptic plasticity - ARC (activity-regulated cytoskeletal protein) and synaptophysin. Gas chromatography-mass spectrometry simultaneously quantified 10 neurosteroid levels in plasma. A history of ethanol drinking and PS exposure produced brain regional and sex differences in the changes in proteins examined as well as in the pattern of neurosteroid levels versus (vs.) values in naïve mice. For instance, P450scc levels were significantly increased only in binge and control female PFC and hippocampus vs. naïve mice. Some neurosteroid levels were significantly altered by binge treatment in both males and females, whereas others were only significantly altered in males. These sexually divergent changes in neurosteroid and protein levels add to evidence for sex differences in the neurochemical systems influenced by traumatic stress and a history of ethanol drinking.

Sex differences in the synergistic effect of prior binge drinking and traumatic stress on subsequent ethanol intake and neurochemical responses in adult C57BL/6J mice.

Alcohol-use disorders (AUDs) are characterized by repeated episodes of binge drinking. Based on reports that exposure to predator odor stress (PS) consistently increases ethanol intake, the present studies examined whether prior binge drinking differentially altered responsivity to PS and subsequent ethanol intake in male and female mice, when compared to mice without prior binge exposure. Initial studies in naïve male and female C57BL/6J mice confirmed that 30-min exposure to dirty rat bedding significantly increased plasma corticosterone (CORT) levels and anxiety-related behavior, justifying the use of dirty rat bedding as PS in the subsequent drinking studies. Next, separate groups of male and female C57BL/6J mice received seven binge ethanol sessions (binge) or drank water (controls), followed by a 1-month period of abstinence. Then, 2-bottle choice ethanol intake (10% or 10E vs. water, 23 h/day) was measured in lickometer chambers for 4 weeks. After baseline intake stabilized, exposure to intermittent PS (2×/week × 2 weeks) significantly enhanced ethanol intake after the 2nd PS in male, but not female, binge mice vs. baseline and vs. the increase in controls. However, in a subgroup of females (with low baselines), PS produced a similar increase in 10E intake in control and binge mice vs. baseline. Analysis of lick behavior determined that the enhanced 10E intake in binge male mice and in the female low baseline subgroup was associated with a significant increase in 10E bout frequency and 10E licks throughout the circadian dark phase. Thus, PS significantly increased 10E intake and had a synergistic interaction with prior binge drinking in males, whereas PS produced a similar significant increase in 10E intake in the low baseline subgroup of binge and control females. Plasma CORT levels were increased significantly in both binge and control animals after PS. CORT levels at 24-h withdrawal from daily 10E intake were highest in the groups with elevated 10E licks (i.e., binge males and control females). At 24-h withdrawal, protein levels of GABAA receptor α1 subunit, corticotropin releasing factor receptor 1, and glucocorticoid receptor in prefrontal cortex (PFC) and hippocampus (HC) were differentially altered in the male and female mice vs. levels in separate groups of age-matched naïve mice, with more changes in HC than in PFC and in females than in males. Importantly, the sexually divergent changes in protein levels in PFC and HC add to evidence for sex differences in the neurochemical systems influenced by stress and binge drinking, and argue for sex-specific pharmacological strategies to treat AUD.

Forecasting academic success through implementation of an online prerequisite review tutorials program for first year pharmacy students.

OBJECTIVE: Online prerequisite review (OPR) tutorials were designed and implemented to reinforce foundational scientific material in order to protect in-class time, foster self-directed learning, and ensure all students have similar baseline knowledge. METHODS: Twenty-one tutorials covering undergraduate prerequisite material were developed by faculty and organized into six core modules, comprising basic biology, chemistry, and physiology topics. A quiz on this material was given on the first day of each course. This score was correlated with the final exam score at course completion. Additional student and faculty feedback was collected through surveys. RESULTS: 2372 quiz-exam pairings were collected over three consecutive fall semesters. A one point increase in the quiz score was associated with a 3.6 point (95% confidence interval 3.1-4.0) higher exam score, as well as a greater probability of passing the exam (P<0.0001). Furthermore, simple linear regression revealed a positive correlation between quiz and exam scores (P<0.0001). Three full years of student survey data revealed an overwhelmingly positive perception of the OPR tutorials, and surveyed faculty reported better use of class time and improved student competency and participation. CONCLUSIONS: Implementation of OPR tutorials may give faculty more efficient use of class time, and their associated quizzes serve as an early indicator for students at-risk of not passing who are candidates for early interventions. Furthermore, the OPR tutorial design gives it great transferability to biomedical post-graduate programs.

Expression of cFos and brain-derived neurotrophic factor in cortex and hippocampus of ethanol-withdrawn male and female rats.

OBJECTIVE: To map areas of brain activation (cFos) alongside changes in levels of brain-derived neurotrophic factor (BDNF) to provide insights into neuronal mechanisms contributing to previously observed sex differences in behavioral measures of ethanol withdrawal (EW). MATERIALS AND METHODS: Immunohistochemical analysis of cFos and BDNF levels using protein-specific antibodies and visualization with nickel-enhanced DAB staining in 3 cortical and 4 hippocampal regions was used to assess EW-induced expression of these proteins. RESULTS: EW male and female rats showed significantly higher levels of cFos expression compared to controls in the hippocampal regions whereas EW OVX rats showed higher levels compared to controls only at 1 day EW in the dentate gyrus. Males expressed higher basal levels of cFos in the CA1 subfield of the hippocampus and in the motor cortex than either intact or OVX female rats. BDNF immunoreactivity was also significantly higher in EW rats compared to that in controls, varying with sex and brain region at 1 and 3 days EW. CONCLUSIONS: Sex-and brain region-specific changes in expression of cFos and BDNF occurring during 1 and 3-day EW, suggest that differential activation and expression of neurotrophins may influence the observed sex differences and support the suggestion that EW is a chronic stressor, eliciting sequential neuronal activation and neurotrophin regulation.

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.

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.

Ethanol-induced loss-of-righting response during ethanol withdrawal in male and female rats: associations with alterations in Arc labeling.

BACKGROUND: There is increasing evidence for relevant sex differences in responses to ethanol. Several investigations have found differences in expression and recovery from ethanol withdrawal (EW) in people and across various animal models. We have found that female rats recover more quickly than male rats and show differential responses to various behavioral assessments and pharmacological challenges during withdrawal. The purpose of this study was to determine whether sex differences in EW behaviors extend to the hypnotic effects of acute ethanol administration. METHODS: We used a repeated measures design to assess duration and latency for loss-of-righting reflex following an acute injection of ethanol (4.2 g/kg; 20% w/v) to pair-fed control or ethanol-withdrawn animals at 1 and 3 days EW in male, female, and ovariectomized female (OVX) rats. We determined protein levels of the activity-regulated cytoskeletal protein (Arc), used as a marker for synaptic activity in glutamatergic synapses, in the motor cortex and prefrontal cortex across these same treatment conditions. RESULTS: Ethanol-withdrawn animals had a reduced ethanol-induced sleep time compared to controls at 1 day EW. Sleep time remained shortened at 3 days EW for males and OVX, but not females. Arc protein levels in motor cortex and preoptic nuclei significantly increased at 1 day EW across all sex conditions, suggestive of an association with the reduced ethanol-induced sleep times during EW. Arc levels increased further for males and OVX, but not females, at the 3 days EW time point. CONCLUSIONS: These findings add further support to sex differences in effects of and responses to ethanol. They suggest that the more rapid recovery from EW for females than males also includes expression of tolerance to the hypnotic effects of ethanol. These sex differences may involve some differential neuroadaptations in glutamatergic signaling.

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.

Sex differences in acoustic startle responses and seizure thresholds between ethanol-withdrawn male and female rats.

AIMS: We have found consistent and significant sex differences in recovery from the increased seizure susceptibility observed during ethanol withdrawal (EW) in our rat model system. The main objective of the present study was to determine if sex differences in EW generalized to an additional behavioral measure startle reactivity. METHODS: Acoustic startle or seizure threshold responses were measured in separate groups of rats at 1 day or 3 days of EW. RESULTS: Both pair-fed control and EW males showed greater increases in acoustic startle responses than either the female or ovariectomized female (OVX) counterparts. There was a selective effect of pregnanolone on acoustic startle in that it reduced peak force of response only at 3 days EW in male rats. Unexpectedly, it modestly increased startle reactivity in control female and OVX rats. Acute treatment with low-dose ethanol trended toward reducing startle responses in control animals, as expected, while generally enhancing startle responses during EW. All sex conditions showed an enhanced startle response during EW following administration of the higher dose of estradiol compared to control animals. Estradiol did not alter seizure thresholds in control animals. However, it was anticonvulsant for males at 3 days EW, females and OVX at 1 day EW. CONCLUSIONS: Observed sex differences in the startle reactivity during EW were consistent with earlier findings comparing EW seizure risk in male and female rats. Responses of OVX suggested that both hormones and differences in brain structures between males and females have a role in these sex differences. Our findings add weight to recommendations that treatment of alcohol withdrawal in humans should consider hormonal status as well as withdrawal time.

Influence of hormonal status on behavioral responses to an acute ethanol challenge during ethanol withdrawal in male and female rats.

We previously reported significant sex differences in ethanol withdrawal (EW) recovery as well as in sensitivity to GABA(A) receptor modulators during EW. The aim of the present study was to determine if hormonal status moderated behavioral responses to an acute ethanol challenge in EW animals comparing two different behaviors. An initial set of experiments explored motor-incoordinating effects of the acute ethanol injection during EW at either 1 day or 3 days of withdrawal. EW male, but not female, rats showed a decrease in coordination compared to controls that persisted through 3 days EW. Female rats displayed tolerance to the motor-incoordinating actions of the acute ethanol challenge at 1 day EW whereas tolerance was more evident in EW male rats at 3 days. In contrast, EW animals generally remained responsive to the anticonvulsant actions of ethanol, irrespective of hormonal status. While EW by itself did not significantly alter seizure latency, duration or severity, it increased seizure-induced mortality especially at 3 days EW. There was some evidence of tolerance to the anticonvulsant effect of the acute ethanol challenge at the lowest dose employed (0.62 g/kg), which varied by sex condition and time of EW. All sex conditions displayed marked sensitivity to the anticonvulsant effects of the ethanol challenge at the two higher doses studied. Overall, ovariectomized females showed the greatest response to the acute ethanol administration. These findings provide additional evidence of a divergence in behavioral responses during EW and suggest that multiple neuroadaptations moderate various responses to ethanol during EW, with minor contributions of hormonal status.

Impact of the hormonal milieu on the neurobiology of alcohol dependence and withdrawal.

Alcoholism, or alcohol dependence, is a complex disorder with withdrawal symptoms that are often problematic for those trying to recover from their dependence. As researchers attempt to elucidate the neurobiological underpinnings of alcohol dependence and withdrawal, it is becoming clear that numerous factors, including the hormonal environment, impact the manifestations of this disorder. Of particular interest is the observation that women have fewer and less severe withdrawal symptoms than do men even though they tend to suffer greater physiological harm from excessive alcohol consumption. In this article, the authors present an overview of their understanding of how gonadal and stress hormones interact with alcohol, which results in differential neurobiological responses between males and females. Thus far, data generated from representative animal models have shown significant differences between the sexes in behavioral responses and neuroadaptations to chronic alcohol consumption and withdrawal. Accumulating evidence suggests that treatment of alcoholism, including withdrawal, should be tailored to the patient's gender and hormonal status.

Impact of sex: determination of alcohol neuroadaptation and reinforcement.

This article represents the proceedings of a symposium at the Research Society on Alcoholism meeting in Santa Barbara, California. The organizers/chairs were Kristine M. Wiren and Deborah A. Finn. Following a brief introduction by Deborah Finn, the presentations were (1) The Importance of Gender in Determining Expression Differences in Mouse Lines Selected for Chronic Ethanol Withdrawal Severity, by Kristine M. Wiren and Joel G. Hashimoto; (2) Sex Differences in Ethanol Withdrawal Involve GABAergic and Stress Systems, by Paul E. Alele and Leslie L. Devaud; (3) The Influence of Sex on Ethanol Consumption and Reward in C57BL/6 Mice, by Kimber L. Price and Lawrence D. Middaugh; and (4) Sex Differences in Alcohol Self-administration in Cynomolgus Monkeys, by Kathleen A. Grant.

Differential effects of mild repeated restraint stress on behaviors and GABA(A) receptors in male and female rats.

We previously reported that the very mild stress of individual housing influenced seizure risk and gamma-amino butyric acid (GABA(A)) receptor activity differentially between male and female rats. The aim of the present set of studies was to assess sex differences in behavioral responses to a more pronounced type of stressor, repeated restraint stress. We also wanted to determine the role of GABA(A) receptors in effects of this stressor. Our data suggest that repeated restraint stress afforded short-term protection against seizure induction in both male and female rats. Moreover, this protection was more persistent in female than male rats. This stress paradigm also elicited a reduction in general activity in male rats, whereas female rats displayed prolonged increased activity following the repeated restraint stress exposure. However, there were limited effects on anxiety-like behaviors, as determined by time spent in the open arms on the elevated plus maze. Sex differences in stress-induced increases in plasma corticosterone levels were observed, which generally correlated with sex differences in behavioral measures. There were no significant effects of the repeated restraint stress exposure on benzodiazepine/GABA(A) receptor density or affinity nor on receptor function. Taken together, these findings provide additional evidence to support the important influences of sex in responding to stress and highlight the need to consider this context when addressing the role of stress in health issues for women and men.

The effects of 17beta-estradiol and ethanol on zinc- or manganese-induced toxicity in SK-N-SH cells.

Serious neurodegenerative disorders are increasingly prevalent in our society and excessive oxidative stress may be a key mediator of neuronal cell death in many of these conditions. A variety of metals, such as manganese and zinc, are essential trace elements but can reach localized toxic concentrations through various disease processes or environmental exposures and have been implicated as having a role in neurodegeneration. Both manganese and zinc exist as bivalent cations and are essential cofactors/activators for numerous enzymes. Evidence suggests one action of these metals, when concentrated beyond physiological levels, may be to inhibit cellular energy production, ultimately leading to increased radical formation. Our studies were undertaken to directly investigate the toxic effects of manganese and zinc in an immortalized neuronal-like cell line (SK-N-SH) by testing interactions with the antioxidant, 17beta-estradiol, and the neurotoxin, ethanol. Employing undifferentiated SK-N-SH cells, we found that these metals caused biphasic effects, enhancing cell proliferation at low doses and inducing cell death at higher doses. Zinc was both more efficacious and more potent than manganese in enhancing growth and in causing cell death. 17beta-Estradiol and ethanol enhanced the proliferative actions of zinc and manganese across a wide concentration range. Furthermore, co-treatment with either 17beta-estradiol or ethanol afforded protection against manganese-, but not zinc-induced toxicity. Finally, combined administration of 17beta-estradiol and ethanol to SK-N-SH cells resulted in both a loss of growth enhancement and protective properties that were observed when these substances were administered individually. We also noted that the toxic effects occurred more rapidly from zinc than manganese exposure. Taken together, these data suggest that oxidative stress likely has a role in cell death resulting from toxic exposure to either zinc or manganese, but there is a difference in the precise mechanism of their effects.

Sex differences in effects of mild chronic stress on seizure risk and GABAA receptors in rats.

Social stress is a common occurrence in our society that can negatively impact health. Therefore, we wanted to study the effects of a mild stressor designed to model social stress on seizure susceptibility and GABAA receptors in male and female rats. The mild chronic stress of individual housing consistently decreased bicuculline (but not pentylenetetrazol, PTZ) seizure thresholds by 10-15% in both sexes. Housing conditions did not alter the anticonvulsant activity of diazepam or ethanol, although the anticonvulsant effect of ethanol was significantly greater against PTZ-induced seizures. Experiments testing the addition of an acute restraint stress unmasked sex differences in seizure induction. The acute stress also selectively decreased the potency of GABA to modulate GABAA receptor-mediated chloride uptake in group-housed females. There were additional sex differences by housing condition for GABAA receptor-gated chloride uptake but no differences in [3H]flunitrazepam binding. We also found significant effects of sex and housing on ethanol-induced increases in corticosterone (CORT) levels. In summary, there were complex and sex-selective effects of mild chronic stress on seizure induction and GABAA receptors. Gaining a better understanding of mechanisms underlying interactions between sex and stress has important implications for addressing health concerns about stress in men and women.

Differential effects of chronic ethanol administration and withdrawal on gamma-aminobutyric acid type A and NMDA receptor subunit proteins in male and female rat brain.

BACKGROUND: Investigations have shown that chronic ethanol exposure results in selective alterations in levels of gamma-aminobutyric acid (GABA)A and NMDA receptor subunits. We previously reported significant sex differences in these chronic ethanol-induced adaptations. Because we have more recently found important sex differences in timing for the development of and recovery from ethanol dependence, we wanted to ascertain whether there were associations between overt expression of withdrawal and neuroadaptations at the level of GABAA and NMDA receptors. METHODS: Western blot analysis was used to assay protein levels for several GABAA and NMDA receptor subunits in rat cerebral cortex and hippocampus by using subunit-selective antibodies. Rats were fed 6% ethanol in a liquid diet with pair-fed controls. Feeding, harvesting of tissue, and Western blot experiments were all conducted while maintaining the paired design. Tissue was harvested after 3 days of ethanol exposure, 9 days of ethanol exposure, or 3 days of ethanol withdrawal after 14 days of liquid diet administration. RESULTS: We again found sex-, subunit-, and brain region-selective effects of ethanol administration and withdrawal for GABAA and NMDA receptors. There was a strong association between increased GABAA receptor alpha4 subunit levels and previously determined withdrawal-induced changes in seizure susceptibility, highlighted by the sex differences in ethanol exposure length required to cause withdrawal signs. In addition, results obtained after 9 days of ethanol administration were in general agreement with previous findings after 14 days of ethanol administration. CONCLUSIONS: These data further support the suggestion that alterations in subunit assembly of GABAA and NMDA receptors may have some mechanistic role in neuroadaptations underlying ethanol dependence and withdrawal. Furthermore, significant sex differences in these adaptations suggest that multiple types of adaptations may be elicited, depending on innate differences in the actions/effects of ethanol.

Sex differences in the central nervous system actions of ethanol.

For many years, researchers have avoided including females in their research because of the poorly understood influences of cycling hormones. However, we are becoming increasingly aware that sex matters, showing that it is important to conduct studies in females as well as males. This review will focus on the central nervous system (CNS) actions of alcohol (ethanol) because we have found significant sex differences in ethanol actions at the molecular as well as the behavioral level. Most recently, in our studies of ethanol dependence and withdrawal, we found that female rats displayed a shorter time for recovery from ethanol withdrawal, assessed by measuring seizure susceptibility. We now report that this finding was confirmed with a second convulsant agent. Moreover, GABAA receptor function was differentially altered in ethanol-withdrawn female compared to male rats. Studies by other investigators have reported additional significant sex differences in ethanol seeking and drinking behaviors and across several measures of ethanol dependence and withdrawal. We are gaining a better understanding of how the actions of ethanol in the CNS overlay sex differences in brain architecture and the hormonal milieu. Therefore, it is not surprising to observe sex-selective effects on cellular and behavioral outcomes from ethanol consumption. While current research is focused on characterizing sex differences in the actions of ethanol, it has not yet reached the point where we can integrate our findings into a unifying concept of how being female differentially regulates CNS responses to ethanol. This is likely a result of the complexity of ethanol actions, involving multiple neurotransmitter systems and responses covering the spectrum from drug seeking behaviors to neuropathological consequences of ethanol misuse. Regardless, the observed sex differences in ethanol withdrawal are noteworthy because they suggest that treatment of alcoholism should be managed differently in women than in men. Finally, it remains important to compare and contrast responses in males and females because recent studies of sex differences in basic physiology have made it clear that being female impacts health and disease.

Effects of ethanol or rimcazole on dizocilpine maleate-induced behaviors in male and female rats.

The current investigation was undertaken to explore further the interactions between ethanol and the phencyclidine analog dizocilpine maleate (MK-801) on behaviors in male and female rats. It was previously found that ethanol dependence conferred cross-tolerance to the behaviorally activating effects of dizocilpine. The current set of studies was designed to assay the interactions between dizocilpine and ethanol in ethanol-naive animals by measuring open field behaviors. I also tested interactions between dizocilpine and rimcazole, a sigma receptor antagonist. In agreement with previous reports, I found significant effects of dizocilpine on several open field behaviors. In general, female rats displayed a lower level of hyperlocomotion and higher level of stereotypies than did male rats. Co-administration of ethanol delayed time to peak hyperlocomotion in male rats. It reduced locomotion in female rats compared with findings for administration of dizocilpine alone. Co-administration of ethanol with dizocilpine increased stereotypies in both sexes. Administration of ethanol increased locomotion to a greater degree in female than in male rats. In contrast, co-administration of rimcazole with dizocilpine had little effect on hyperlocomotion in male rats while increasing levels in female rats. Rimcazole increased dizocilpine-induced stereotypies to a greater extent in male than in female rats. Results of receptor-binding studies revealed small differences for cerebral cortical sigma receptors between male and female rats. Dizocilpine was unable to compete for sigma receptor-binding sites. This is in contrast to phencyclidine, which acts at both N-methyl-D-aspartate (NMDA) and sigma receptors. These findings extend previous evidence of interactions between ethanol and dizocilpine, but highlight that responses vary by measure, sex, and length of ethanol exposure. In addition, findings from the current study uncovered sex-selective interactions between dizocilpine and a sigma receptor ligand, providing further evidence for complex actions and interactions of this noncompetitive NMDA receptor antagonist with multiple sites in brain.