Stress and gonadal steroid influences on alcohol drinking and withdrawal, with focus on animal models in females.

Sexually dimorphic effects of alcohol, following binge drinking, chronic intoxication, and withdrawal, are documented at the level of the transcriptome and in behavioral and physiological responses. The purpose of the current review is to update and to expand upon contributions of the endocrine system to alcohol drinking and withdrawal in females, with a focus on animal models. Steroids important in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes, the reciprocal interactions between these axes, the effects of chronic alcohol use on steroid levels, and the genomic and rapid membrane-associated effects of steroids and neurosteroids in models of alcohol drinking and withdrawal are described. Importantly, comparison between males and females highlight some divergent effects of sex- and stress-steroids on alcohol drinking- and withdrawal-related behaviors, and the distinct differences in response emphasize the importance of considering sex in the development of novel pharmacotherapies for the treatment of alcohol use disorder.

Effects of sleep disruption on stress, nigrostriatal markers, and behavior in a chronic/progressive MPTP male mouse model of parkinsonism.

Sleep complaints are an early clinical symptom of neurodegenerative disorders. Patients with Parkinson's disease (PD) experience sleep disruption (SD). The objective of this study was to determine if preexisting, chronic SD leads to a greater loss of tyrosine hydroxylase (TH) within the striatum and the substantia nigra following chronic/progressive exposure with the neurotoxin, 1-methyl-2-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male mice underwent chronic SD for 4 weeks, then injected with vehicle (VEH) or increasing doses of MPTP for 4 weeks. There was a significant decrease in the plasma corticosterone levels in the MPTP group, an increase in the SD group, and a return to the VEH levels in the SD+MPTP group. Protein expression levels for TH in the striatum (terminals) and substantia nigra pars compacta (dopamine [DA] cell counts) revealed up to a 78% and 38% decrease, respectively, in the MPTP and SD+MPTP groups compared to their relevant VEH and SD groups. DA transporter protein expression increased in the striatum in the MPTP versus VEH group and in the SN/midbrain between the SD+MPTP and the VEH group. There was a main effect of MPTP on various gait measures (e.g., braking) relative to the SD or VEH groups. In the SD+MPTP group, there were no differences compared to the VEH group. Thus, SD, prior to administration of MPTP, has effects on serum corticosterone and gait but more importantly does not potentiate greater loss of TH within the nigrostriatal pathway compared to the MPTP group, suggesting that in PD patients with SD, there is no exacerbation of the DA cell loss.

Dynamic Adaptation in Neurosteroid Networks in Response to Alcohol.

The term neurosteroid refers to rapid membrane actions of steroid hormones and their derivatives that can modulate physiological functions and behavior via their interactions with ligand-gated ion channels. This chapter will highlight recent advances pertaining to the modulatory effects of a select group of neurosteroids that are primarily potent positive allosteric modulators of γ-aminobutyric acidA receptors (GABAARs). Nanomolar concentrations of neurosteroids, which occur in vivo, potentiate phasic and tonic forms of GABAAR-mediated inhibition, indicating that both synaptic and extrasynaptic GABAARs possess sensitivity to neurosteroids and contribute to the overall ability of neurosteroids to modulate central nervous system excitability. Common effects of alcohol and neurosteroids at GABAARs have stimulated research on the ability of neurosteroids to modulate alcohol's acute and chronic effects. Background on neurosteroid pharmacology and biosynthetic enzymes will be provided as it relates to experimental findings. Data will be summarized on alcohol and neurosteroid interactions across neuroanatomical regions and models of intoxication, consumption, dependence, and withdrawal. Evidence supports independent regulation of neurosteroid synthesis between periphery and brain as well as across brain regions following acute alcohol administration and during withdrawal. Local mechanisms for fine-tuning neuronal excitability via manipulation of neurosteroid synthesis exert predicted behavioral and electrophysiological responses on GABAAR-mediated inhibition. Collectively, targeting neurosteroidogenesis may be a beneficial treatment strategy for alcohol use disorders.

Alcohol Suppresses Tonic GABAA Receptor Currents in Cerebellar Granule Cells in the Prairie Vole: A Neural Signature of High-Alcohol-Consuming Genotypes.

BACKGROUND: Evidence indicates that the cerebellum plays a role in genetic predilection to excessive alcohol (ethanol [EtOH]) consumption in rodents and humans, but the molecular mechanisms mediating such predilection are not understood. We recently determined that EtOH has opposite actions (enhancement or suppression) on tonic GABAA receptor (GABAA R) currents in cerebellar granule cells (GCs) in low- and high-EtOH-consuming rodents, respectively, and proposed that variation in GC tonic GABAA R current responses to EtOH contributes to genetic variation in EtOH consumption phenotype. METHODS: Voltage-clamp recordings of GCs in acutely prepared slices of cerebellum were used to evaluate the effect of EtOH on GC tonic GABAA R currents in another high-EtOH-consuming rodent, prairie voles (PVs). RESULTS: EtOH (52 mM) suppressed the magnitude of the tonic GABAA R current in 57% of cells, had no effect in 38% of cells, and enhanced the tonic GABAA R current in 5% of cells. This result is similar to GCs from high-EtOH-consuming C57BL/6J (B6) mice, but it differs from the enhancement of tonic GABAA R currents by EtOH in low-EtOH-consuming DBA/2J (D2) mice and Sprague Dawley (SD) rats. EtOH suppression of tonic GABAA R currents was not affected by the sodium channel blocker, tetrodotoxin (500 nM), and was independent of the frequency of phasic GABAA R-mediated currents, suggesting that suppression is mediated by postsynaptic actions on GABAA Rs, rather than a reduction of GABA release. Finally, immunohistochemical analysis of neuronal nitric oxide synthase (nNOS; which can mediate EtOH enhancement of GABA release) demonstrated that nNOS expression in the GC layer of PV cerebellum was similar to the levels seen in B6 mice, both being significantly reduced relative to D2 mice and SD rats. CONCLUSIONS: Combined, these data highlight the GC GABAA R response to EtOH in another species, the high-EtOH-consuming PV, which correlates with EtOH consumption phenotype and further implicates the GC GABAA R system as a contributing mechanism to high EtOH consumption.

Tigecycline Reduces Ethanol Intake in Dependent and Nondependent Male and Female C57BL/6J Mice.

BACKGROUND: The chronic intermittent ethanol (CIE) paradigm is valuable for screening compounds for efficacy to reduce drinking traits related to alcohol use disorder (AUD), as it measures alcohol consumption and preference under physical dependence conditions. Air control-treated animals allow simultaneous testing of similarly treated, nondependent animals. As a consequence, we used CIE to test the hypothesis that tigecycline, a semisynthetic tetracycline similar to minocycline and doxycycline, would reduce alcohol consumption regardless of dependence status. METHODS: Adult C57BL/6J female and male mice were tested for tigecycline efficacy to reduce ethanol (EtOH) consumption using a standard CIE paradigm. The ability of tigecycline to decrease 2-bottle choice of 15% EtOH (15E) versus water intake in dependent (CIE vapor) and nondependent (air-treated) male and female mice was tested after 4 cycles of CIE vapor or air exposure using a within-subjects design and a dose-response. Drug doses of 0, 40, 60, 80, and 100 mg/kg in saline were administered intraperitoneally (0.01 ml/g body weight) and in random order, with a 1-hour pretreatment time. Baseline 15E intake was re-established prior to administration of subsequent injections, with a maximum of 2 drug injections tested per week. RESULTS: Tigecycline was found to effectively reduce high alcohol consumption in both dependent and nondependent female and male mice. CONCLUSIONS: Our data suggest that tigecycline may be a promising drug with novel pharmacotherapeutic characteristics for the treatment of mild-to-severe AUD in both sexes.

Sex and the Lab: An Alcohol-Focused Commentary on the NIH Initiative to Balance Sex in Cell and Animal Studies.

In May 2014, Dr. Francis Collins, the director of U.S. National Institutes of Health (NIH), and Dr. Janine Clayton, the director of the U.S. National Institutes of Health Office of Research on Women's Health, published a commentary in the journal Nature announcing new policies to ensure that preclinical research funded by the NIH considers both males and females. While these policies are still developing, they have already generated great interest by the scientific community and triggered both criticism and applause. This review provides a description and interpretation of the NIH guidelines, and it traces the history that led to their implementation. As expected, this NIH initiative generated some anxiety in the scientific community. The use of female animals in the investigation of basic mechanisms is perceived to increase variability in the results, and the use of both sexes has been claimed to slow the pace of scientific discoveries and to increase the cost at a time characterized by declining research support. This review discusses issues related to the study of sex as a biological variable (SABV) in alcohol studies and provides examples of how researchers have successfully addressed some of them. A practical strategy is provided to include both sexes in biomedical research while maintaining control of the research direction. The inclusion of sex as an important biological variable in experimental design, analysis, and reporting of preclinical alcohol research is likely to lead to a better understanding of alcohol pharmacology and the development of alcohol use disorder, may promote drug discovery for new pharmacotherapies by increasing scientific rigor, and may provide clinical benefit to women's health. This review aims to promote the understanding of the NIH's SABV guidelines and to provide alcohol researchers with a theoretical and practical framework for working with both sexes in preclinical research.

Effective Reduction of Acute Ethanol Withdrawal by the Tetracycline Derivative, Tigecycline, in Female and Male DBA/2J Mice.

BACKGROUND: Alcohol use disorder (AUD) is a spectrum disorder characterized by mild to severe symptoms, including potential withdrawal signs upon cessation of consumption. Approximately five hundred thousand patients with AUD undergo clinically relevant episodes of withdrawal annually (New Engl J Med, 2003, 348, 1786). Recent evidence indicates potential for drugs that alter neuroimmune pathways as new AUD therapies. We have previously shown the immunomodulatory drugs, minocycline and tigecycline, were effective in reducing ethanol (EtOH) consumption in both the 2-bottle choice and drinking-in-the-dark paradigms. Here, we test the hypothesis that tigecycline, a tetracycline derivative, will reduce the severity of EtOH withdrawal symptoms in a common acute model of alcohol withdrawal (AWD) using a single anesthetic dose of EtOH in seizure sensitive DBA/2J (DBA) mice. METHODS: Naïve adult female and male DBA mice were given separate injections of 4 g/kg i.p. EtOH with vehicle or tigecycline (0, 20, 40, or 80 mg/kg i.p.). The 80 mg/kg dose was tested at 3 time points (0, 4, and 7 hours) post EtOH treatment. Handling-induced convulsions (HICs) were measured before and then over 12 hours following EtOH injection. HIC scores and areas under the curve were tabulated. In separate mice, blood EtOH concentrations (BECs) were measured at 2, 4, and 7 hours postinjection of 4 g/kg i.p. EtOH in mice treated with 0 and 80 mg/kg i.p. tigecycline. RESULTS: AWD symptom onset, peak magnitude, and overall HIC severity were reduced by tigecycline drug treatment compared to controls. Tigecycline treatment was effective regardless of timing throughout AWD, with earlier treatment showing greater efficacy. Tigecycline showed a dose-responsive reduction in acute AWD convulsions, with no sex differences in efficacy. Importantly, tigecycline did not affect BECs over a time course of elimination. CONCLUSIONS: Tigecycline effectively reduced AWD symptoms in DBA mice at all times and dosages tested, making it a promising lead compound for development of a novel pharmacotherapy for AWD. Further studies are needed to determine the mechanism of tigecycline action.

Null mutation of 5α-reductase type I gene alters ethanol consumption patterns in a sex-dependent manner.

The neuroactive steroid allopregnanolone (ALLO) is a positive modulator of GABAA receptors, and manipulation of neuroactive steroid levels via injection of ALLO or the 5α-reductase inhibitor finasteride alters ethanol self-administration patterns in male, but not female, mice. The Srd5a1 gene encodes the enzyme 5α-reductase-1, which is required for the synthesis of ALLO. The current studies investigated the influence of Srd5a1 deletion on voluntary ethanol consumption in male and female wildtype (WT) and knockout (KO) mice. Under a continuous access condition, 6 and 10 % ethanol intake was significantly greater in KO versus WT females, but significantly lower in KO versus WT males. In 2-h limited access sessions, Srd5a1 deletion retarded acquisition of 10 % ethanol intake in female mice, but facilitated it in males, versus respective WT mice. The present findings demonstrate that the Srd5a1 gene modulates ethanol consumption in a sex-dependent manner that is also contingent upon ethanol access condition and concentration.

Sex Differences in Ethanol's Anxiolytic Effect and Chronic Ethanol Withdrawal Severity in Mice with a Null Mutation of the 5α-Reductase Type 1 Gene.

Manipulation of endogenous levels of the GABAergic neurosteroid allopregnanolone alters sensitivity to some effects of ethanol. Chronic ethanol withdrawal decreases activity and expression of 5α-reductase-1, an important enzyme in allopregnanolone biosynthesis encoded by the 5α-reductase-1 gene (Srd5a1). The present studies examined the impact of Srd5a1 deletion in male and female mice on several acute effects of ethanol and on chronic ethanol withdrawal severity. Genotype and sex did not differentially alter ethanol-induced hypothermia, ataxia, hypnosis, or metabolism, but ethanol withdrawal was significantly lower in female versus male mice. On the elevated plus maze, deletion of the Srd5a1 gene significantly decreased ethanol's effect on total entries versus wildtype (WT) mice and significantly decreased ethanol's anxiolytic effect in female knockout (KO) versus WT mice. The limited sex differences in the ability of Srd5a1 genotype to modulate select ethanol effects may reflect an interaction between developmental compensations to deletion of the Srd5a1 gene with sex hormones and levels of endogenous neurosteroids.

The neurosteroid allopregnanolone impairs object memory and contextual fear memory in male C57BL/6J mice.

Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: (i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.

Contribution of P2X4 receptors to ethanol intake in male C57BL/6 mice.

P2X receptors (P2XRs) are a family of cation-permeable ligand-gated ion channels activated by synaptically released extracellular adenosine 5'-triphosphate. The P2X4 subtype is abundantly expressed in the central nervous system and is sensitive to low intoxicating ethanol concentrations. Genetic meta-analyses identified the p2rx4 gene as a candidate gene for innate alcohol intake and/or preference. The current study used mice lacking the p2rx4 gene (knockout, KO) and wildtype (WT) C57BL/6 controls to test the hypothesis that P2X4Rs contribute to ethanol intake. The early acquisition and early maintenance phases of ethanol intake were measured with three different drinking procedures. Further, we tested the effects of ivermectin (IVM), a drug previously shown to reduce ethanol's effects on P2X4Rs and to reduce ethanol intake and preference, for its ability to differentially alter stable ethanol intake in KO and WT mice. Depending on the procedure and the concentration of the ethanol solution, ethanol intake was transiently increased in P2X4R KO versus WT mice during the acquisition of 24-h and limited access ethanol intake. IVM significantly reduced ethanol intake in P2X4R KO and WT mice, but the degree of reduction was 50 % less in the P2X4R KO mice. Western blot analysis identified significant changes in γ-aminobutyric acidA receptor α1 subunit expression in brain regions associated with the regulation of ethanol behaviors in P2X4R KO mice. These findings add to evidence that P2X4Rs contribute to ethanol intake and indicate that there is a complex interaction between P2X4Rs, ethanol, and other neurotransmitter receptor systems.

The Effect of mGluR5 Antagonism During Binge Drinkingon Subsequent Ethanol Intake in C57BL/6J Mice: Sex- and Age-Induced Differences.

BACKGROUND: Binge ethanol (EtOH) intake during adolescence leads to an array of behavioral and cognitive consequences including elevated intake of EtOH during adulthood, with female mice showing greater susceptibility than males. Administration of the metabotropic glutamate receptor 5 (mGluR5) antagonist 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) has been shown to reduce EtOH self-administration in adult male mice, but little is known about its effect on female and adolescent mice. METHODS: MTEP (0, 10, 20 mg/kg, i.p.) was repeatedly administered to female and male, adult and adolescent C57BL/6J mice during binge sessions using the scheduled high alcohol consumption paradigm. Next, we assessed whether MTEP administration during binge altered the subsequent 24-hour EtOH intake following a period of abstinence. Finally, we investigated whether MTEP administration during binge followed by an abstinence period altered mRNA of glutamatergic genes within the nucleus accumbens of female mice. RESULTS: MTEP significantly decreased binge EtOH intake in all mice, but only female mice exhibited altered subsequent 24-hour EtOH intake. Interestingly, the alteration in subsequent EtOH intake in female animals was age dependent, with adolescent exposure to MTEP during binge decreasing 24-hour intake and adult exposure to MTEP during binge increasing 24-hour intake. Finally, while there were no effects of MTEP pretreatment on the genes examined, there was a robust age effect found during analysis of mGluR1 (Grm1), mGluR5 (Grm5), the NR2A subunit of the NMDA receptor (Grin2a), phosphatidylinositol 3-kinase (Pik3r1), mammalian target of rapamycin (Mtor), and extracellular signal-regulated kinase (Mapk1) mRNA, with adolescent female animals having lower expression than their adult counterparts. CONCLUSIONS: Collectively, the present findings add to existing evidence implicating the contribution of long-term effects of adolescent binge drinking to enhance alcohol abuse in adulthood, while suggesting that mGluR5 antagonism may not be the best pharmacotherapy to treat binge alcohol consumption in female and adolescent animals.

Traumatic stress-enhanced alcohol drinking: Sex differences and animal model perspectives.

Purpose of review: Stress is associated with alcohol drinking, and epidemiological studies document the comorbidity of alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD), with higher comorbid prevalence in females than in males. The aim of this paper is to highlight information related to sex differences in stress-enhanced alcohol drinking from clinical studies and from preclinical studies utilizing an animal model of traumatic stress. Recent findings: Stress is associated with alcohol drinking and relapse in males and females, but there are sex differences in the alcohol-related adaptation of stress pathways and in the association of different prefrontal regions with stress-induced anxiety. The predator stress model of traumatic stress produced enhanced alcohol drinking in a subgroup of stress-sensitive male and female animals, which could be associated with sex and subgroup differences in stress axis responsivity, behavioral responses to predator odors, and epigenetic mechanisms engaged by traumatic experiences. Summary: While additional studies in females are necessary, existing clinical and preclinical evidence suggests that biological mechanisms underlying stress-enhanced drinking likely differ between males and females. Thus, effective treatment strategies may differ between the sexes.

Stress-enhanced ethanol drinking does not increase sensitivity to the effects of a CRF-R1 antagonist on ethanol intake in male and female mice.

Research confirms that stress is associated with alcohol drinking and relapse in males and females and that there are sex differences in the alcohol-related adaptations of stress pathways. The predator stress (PS) model of traumatic stress produces an increase in alcohol drinking or self-administration in a subpopulation of rodents, so it is utilized as an animal model of comorbid alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD). Previous work determined that sensitivity to PS-enhanced drinking produced sex differences in proteins related to stress-regulating systems in the medial prefrontal cortex and hippocampus. The present studies examined whether male and female C57BL/6J mice differ in sensitivity to the ability of the corticotropin releasing factor receptor 1 antagonist CP-376395 to decrease PS-enhanced drinking. In control studies, CP-376395 doses of 5, 10, and 20 mg/kg dose-dependently decreased 4-hour ethanol drinking. Next, CP-376395 doses of 5 and 10 mg/kg were tested for effects on ethanol drinking in mice with differential sensitivity to PS-enhanced drinking. Subgroups of "Sensitive" and "Resilient" male and female mice were identified based on changes in ethanol intake in an unrestricted access ethanol drinking procedure following four exposures to PS (dirty rat bedding). During the first 2 hours post-injection of CP-376395, both doses significantly decreased ethanol licks versus vehicle in the females, with no significant interaction between subgroups, whereas the 10 mg/kg dose significantly decreased ethanol licks versus vehicle in the "Resilient" males. Thus, sensitivity to the suppressive effect of CP-376395 on stress-induced ethanol intake was greater in females versus males, whereas sensitivity and resilience to PS-enhanced drinking produced differential sensitivity to the ability of CP-376395 to decrease ethanol drinking only in male mice. Our results argue against greater efficacy of CRF-R1's ability to decrease ethanol intake in subjects with traumatic stress-enhanced ethanol drinking.

Pharmacological insights into the role of P2X4 receptors in behavioural regulation: lessons from ivermectin.

Purinergic ionotropic P2X receptors are a family of cation-permeable channels that bind extracellular adenosine 5'-triphosphate. In particular, convergent lines of evidence have recently highlighted P2X(4) receptors as a potentially critical target in the regulation of multiple nervous and behavioural functions, including pain, neuroendocrine regulation and hippocampal plasticity. Nevertheless, the role of the P2X(4) receptor in behavioural organization remains poorly investigated. To study the effects of P2X(4) activation, we tested the acute effects of its potent positive allosteric modulator ivermectin (IVM, 2.5-10 mg/kg i.p.) on a broad set of paradigms capturing complementary aspects of perceptual, emotional and cognitive regulation in mice. In a novel open field, IVM did not induce significant changes in locomotor activity, but increased the time spent in the peripheral zone. In contrast, IVM produced anxiolytic-like effects in the elevated plus maze and marble burying tasks, as well as depression-like behaviours in the tail-suspension and forced swim tests. The agent induced no significant behavioural changes in the conditioned place preference test and in the novel object recognition task. Finally, the drug induced a dose-dependent decrease in sensorimotor gating, as assessed by pre-pulse inhibition (PPI) of the acoustic startle reflex. In P2X(4) knockout mice, the effects of IVM in the open field and elevated plus maze were similar to those observed in wild type mice; conversely, the drug significantly increased startle amplitude and failed to reduce PPI. Taken together, these results suggest that P2X(4) receptors may play a role in the regulation of sensorimotor gating.

Recent advances in the discovery and preclinical testing of novel compounds for the prevention and/or treatment of alcohol use disorders.

Alcohol abuse and dependence have a staggering socioeconomic impact, yet current therapeutic strategies are largely inadequate to treat these disorders. Thus, the development of new strategies that can effectively prevent alcohol use disorders (AUDs) is of paramount importance. Currently approved medications attempt to deter alcohol intake by blocking ethanol metabolism or by targeting the neurochemical systems downstream of the cascades leading to craving and dependence. Unfortunately, these medications have provided only limited success as indicated by the continued high rates of alcohol abuse and alcoholism. The lack of currently available effective treatment strategies is highlighted by the urgent call by the NIAAA to find new and paradigm-changing therapeutics to either prevent or treat alcohol-related problems. This mini-review highlights recent findings from 4 laboratories with a focus on compounds that have the potential to be novel therapeutic agents that can be developed for the prevention and/or treatment of AUDs.

Local changes in neurosteroid levels in the substantia nigra reticulata and the ventral tegmental area alter chronic ethanol withdrawal severity in male withdrawal seizure-prone mice.

BACKGROUND: Allopregnanolone (ALLO) is a potent positive modulator of γ-aminobutyric acidA receptors (GABAA Rs) that affects ethanol (EtOH) withdrawal. Finasteride (FIN), a 5α-reductase inhibitor that blocks the formation of ALLO and other GABAergic neurosteroids, alters EtOH sensitivity. Recently, we found that Withdrawal Seizure-Prone mice from the first genetic replicate (WSP-1) exhibited behavioral tolerance to the anticonvulsant effect of intrahippocampal ALLO during EtOH withdrawal and that intrahippocampal FIN significantly increased EtOH withdrawal severity. The purpose of this study was to determine whether neurosteroid manipulations in the substantia nigra reticulata (SNR) and ventral tegmental area (VTA) produced effects during EtOH withdrawal comparable to those seen with intrahippocampal ALLO and FIN. METHODS: Male WSP-1 mice were surgically implanted with bilateral guide cannulae aimed at the SNR or VTA at 2 weeks prior to EtOH vapor or air exposure for 72 hours. Initial studies examined the anticonvulsant effect of a single ALLO infusion (0, 100, or 400 ng/side) at a time corresponding to peak withdrawal in the air- and EtOH-exposed mice. Separate studies examined the effect of 4 FIN infusions (0 or 10 µg/side/d) during the development of physical dependence on the expression of EtOH withdrawal. RESULTS: ALLO infusion exerted a potent anticonvulsant effect in EtOH-naïve mice, but a diminished anticonvulsant effect during EtOH withdrawal. Administration of FIN into the SNR exerted a delayed proconvulsant effect in EtOH-naïve mice, whereas infusion into the VTA increased EtOH withdrawal duration. CONCLUSIONS: Activation of local GABAA Rs in the SNR and VTA via ALLO infusion is sufficient to exert an anticonvulsant effect in naïve mice and to produce behavioral tolerance to the anticonvulsant effect of ALLO infusion during EtOH withdrawal. Thus, EtOH withdrawal reduced sensitivity of GABAA Rs to GABAergic neurosteroids in 2 neuroanatomical substrates within the basal ganglia in WSP-1 male mice.

Differential c-Fos Response in Neurocircuits Activated by Repeated Predator Stress in Male and Female C57BL/6J Mice with Stress Sensitive or Resilient Alcohol Intake Phenotypes.

Comorbidity of post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) worsens the prognosis for each of these individual disorders. The current study aimed to identify neurocircuits potentially involved in regulation of PTSD-AUD comorbidity by mapping expression of c-Fos in male and female C57BL/6J mice following repeated predator stress (PS), modeled by exposure to dirty rat bedding. In experiment 1, the levels of c-Fos in the paraventricular nucleus of the hypothalamus (PVH) and the nucleus accumbens shell were higher after the second PS vs the first PS, indicating a sensitized response to this stressor. Additional brain regions showed varied sex-dependent and independent regulation by the two consecutive PS exposures. In experiment 2, mice that increased voluntary alcohol consumption following four exposures to PS (Sensitive subgroup) showed higher c-Fos induction in the PVH, piriform cortex and ventromedial hypothalamus than mice that decreased consumption following these exposures (Resilient subgroup). In contrast to these brain regions, c-Fos was higher in the anterior olfactory nucleus of Resilient vs Sensitive mice. Taken together, these data demonstrate that repeated PS exposure and voluntary alcohol consumption increase neuronal activity across neurocircuits in which specific components depend on the vulnerability of individual mice to these stressors. Increased PVH activity observed across both experiments suggests this brain area as a potential mediator of PS-induced increases in alcohol consumption. Future investigations of specific neuronal populations within the PVH activated by PS, and manipulation of these specific neuronal populations, could improve our understanding of the mechanisms leading to PTSD-AUD comorbidity.

Corticosterone Levels and Glucocorticoid Receptor Gene Expression in High Drinking in the Dark Mice and Their Heterogeneous Stock (HS/NPT) Founder Line.

The High Drinking in the Dark (HDID-1) line of mice has been selectively bred for achieving high blood alcohol levels (BALs) in the Drinking in the Dark task, a model of binge-like drinking. Recently, we determined that glucocorticoid receptor (GR) antagonism with either mifepristone or CORT113176 (a selective GR antagonist) reduced binge-like ethanol intake in the HDID-1 mice, but not in their founder line, HS/NPT. Here, we examined whether the selection process may have altered glucocorticoid functioning by measuring (1) plasma corticosterone levels and (2) expression of the genes encoding GR (Nr3c1) and two of its chaperone proteins FKBP51 and FKBP52 (Fkbp5 and Fkbp4) in the brains (nucleus accumbens, NAc) of HDID-1 and HS/NPT mice. We observed no genotype differences in baseline circulating corticosterone levels. However, HDID-1 mice exhibited a greater stimulated peak corticosterone response to an IP injection (of either ethanol or saline) relative to their founder line. We further observed reduced basal expression of Fkbp4 and Nr3c1 in the NAc of HDID-1 mice relative to HS/NPT mice. Finally, HDID-1 mice exhibited reduced Fkbp5 expression in the NAc relative to HS/NPT mice following an injection of 2 g/kg ethanol. Together, these data suggest that selective breeding for high BALs may have altered stress signaling in the HDID-1 mice, which may contribute to the observed selective efficacy of GR antagonism in reducing binge-like ethanol intake in HDID-1, but not HS/NPT mice. These data have important implications for the role that stress signaling plays in the genetic risk for binge drinking.

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.