Alcohol use disorder (AUD) and major depressive disorder (MDD) are prevalent, debilitating, and highly comorbid disorders. The molecular changes that underlie their comorbidity are beginning to emerge. For example, recent evidence showed that acute ethanol exposure produces rapid antidepressant-like biochemical and behavioral responses. Both ethanol and fast-acting antidepressants block N-methyl-D-aspartate receptor (NMDAR) activity, leading to synaptic changes and long-lasting antidepressant-like behavioral effects. We used RNA sequencing to analyze changes in the synaptic transcriptome after acute treatment with ethanol or the NMDAR antagonist, Ro 25-6981. Ethanol and Ro 25-6981 induced differential, independent changes in gene expression. In contrast with gene-level expression, ethanol and Ro 25-6981 produced overlapping changes in exons, as measured by analysis of differentially expressed exons (DEEs). A prominent overlap in genes with DEEs indicated that changes in exon usage were important for both ethanol and Ro 25-6981 action. Structural modeling provided evidence that ethanol-induced exon expression in the NMDAR1 amino-terminal domain could induce conformational changes and thus alter NMDAR function. These findings suggest that the rapid antidepressant effects of ethanol and NMDAR antagonists reported previously may depend on synaptic exon usage rather than gene expression.

The agranular insular cortex (AIC) has recently been investigated by the alcohol field because of its connectivity to and modulatory control over limbic and brainstem regions implicated in alcohol use disorder (AUD), and because it has shown involvement in animal models of alcohol drinking. Despite evidence of AIC involvement in AUD, there has not yet been an examination of whether ethanol modulatesglutamatergic and γ-amino-butyric acid (GABA)ergic synaptic transmission and plasticity in the AIC. Characterizing how the synaptictransmission and plasticity states of AIC cortical processing neurons are modulated by acute ethanol will likely reveal the molecular targets by which chronic ethanol alters AIC function as alcohol drinking transitions from controlled to problematic. Therefore, we collected brain slices from ethanol-naïve adult male mice, obtained whole-cell recording configuration in layer 2/3 AIC pyramidal neurons, and bath-applied ethanolat pharmacologically relevant concentrations during electrophysiological assays of glutamatergic and GABAergic synaptic transmission and plasticity. We found that ethanol inhibited electrically evoked N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory post-synapticcurrents (EPSCs) in a concentration-related fashion, and had little effect on evoked α-amino-3-hydrox-5-methylisoxazole-4-propionic acid-type receptor (AMPAR)-mediated EPSCs. Ethanol had no effect on spontaneous excitatory post-synaptic currents (sEPSCs) or inhibitory GABA_AR-mediated post-synaptic currents (sIPSCs). We found that synaptic conditioning (low-frequency stimulation for 15 min at 1 Hz) induced a form of long-term depression (LTD) of evoked AMPAR-mediated EPSCs. The ability to induce LTD was inhibited by a non-selective NMDAR antagonist (DL-2-amino-5-phosphonovaleric acid), and also by acute, intoxicating concentrations of ethanol. Taken together these data suggest that the glutamate, but not GABA system in the AIC is uniquely sensitive to ethanol, and that in particular NMDAR-mediatedprocesses in the AIC may be disrupted by pharmacologically relevant concentrations of ethanol.

The alcohol research field has amassed an impressive number of gene expression datasets spanning key brain areas for addiction, species (humans as well as multiple animal models), and stages in the addiction cycle (binge/intoxication, withdrawal/negative effect, and preoccupation/anticipation). These data have improved our understanding of the molecular adaptations that eventually lead to dysregulation of brain function and the chronic, relapsing disorder of addiction. Identification of new medications to treat alcohol use disorder (AUD) will likely benefit from the integration of genetic, genomic, and behavioral information included in these important datasets. Systems pharmacology considers drug effects as the outcome of the complex network of interactions a drug has rather than a single drug-molecule interaction. Computational strategies based on this principle that integrate gene expression signatures of pharmaceuticals and disease states have shown promise for identifying treatments that ameliorate disease symptoms (called in silico gene mapping or connectivity mapping). In this review, we suggest that gene expression profiling for in silico mapping is critical to improve drug repurposing and discovery for AUD and other psychiatric illnesses. We highlight studies that successfully apply gene mapping computational approaches to identify or repurpose pharmaceutical treatments for psychiatric illnesses. Furthermore, we address important challenges that must be overcome to maximize the potential of these strategies to translate to the clinic and improve healthcare outcomes.

BACKGROUND:

Transcriptional differences between heterogeneous stock mice and high drinking-in-the-dark selected mouse lines have previously been described based on microarray technology coupled with network-based analysis. The network changes were reproducible in 2 independent selections and largely confined to 2 distinct network modules; in contrast, differential expression appeared more specific to each selected line. This study extends these results by utilizing RNA-Seq technology, allowing evaluation of the relationship between genetic risk and transcription of noncoding RNA (ncRNA); we additionally evaluate sex-specific transcriptional effects of selection.

METHODS:

Naïve mice (N = 24/group and sex) were utilized for gene expression analysis in the ventral striatum; the transcriptome was sequenced with the Illumina HiSeq platform. Differential gene expression and the weighted gene co-expression network analysis were implemented largely as described elsewhere, resulting in the identification of genes that change expression level or (co)variance structure.

RESULTS:

Across both sexes, we detect selection effects on the extracellular matrix and synaptic signaling, although the identity of individual genes varies. A majority of nc RNAs cluster in a single module of relatively low density in both the male and female network. The most strongly differentially expressed transcript in both sexes was Gm22513, a small nuclear RNA with unknown function. Associated with selection, we also found a number of network hubs that change edge strength and connectivity. At the individual gene level, there are many sex-specific effects; however, at the annotation level, results are more concordant.

CONCLUSIONS:

In addition to demonstrating sex-specific effects of selection on the transcriptome, the data point to the involvement of extracellular matrix genes as being associated with the binge drinking phenotype.

Transcriptome-based drug discovery has identified new treatments for some complex diseases, but has not been applied to alcohol use disorder (AUD) or other psychiatric diseases, where there is a critical need for improved pharmacotherapies. High Drinking in the Dark (HDID-1) mice are a genetic model of AUD risk that have been selectively bred (from the HS/Npt line) to achieve intoxicating blood alcohol levels (BALs) after binge-like drinking. We compared brain gene expression of HDID-1 and HS/Npt mice, to determine a molecular signature for genetic risk for high intensity, binge-like drinking. Using multiple computational methods, we queried LINCS-L1000 (Library of Integrated Network-Based Cellular Signatures), a database containing gene expression signatures of thousands of compounds, to predict candidate drugs with the greatest potential to decrease alcohol consumption. Our analyses predicted novel compounds for testing, many with anti-inflammatory properties, providing further support for a neuroimmune mechanism of excessive alcohol drinking. We validated the top 2 candidates in vivo as a proof-of-concept. Terreic acid (a Bruton's tyrosine kinase inhibitor) and pergolide (a dopamine and serotonin receptor agonist) robustly reduced alcohol intake and BALs in HDID-1 mice, providing the first evidence for transcriptome-based drug discovery to target an addiction trait. Effective drug treatments for many psychiatric diseases are lacking, and the emerging tools and approaches outlined here offer researchers studying complex diseases renewed opportunities to discover new or repurpose existing compounds and expedite treatment options.

While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.

Background Although pharmacotherapies are available for alcohol (EtOH) or tobacco use disorders individually, it may be possible to develop a single pharmacotherapy to treat heavy drinking tobacco smokers by capitalizing on the commonalities in their mechanisms of action. Methods Female alcohol-preferring (P) rats were trained for EtOH drinking and nicotine self-administration in two phases: (1) EtOH alone (0 vs. 15% EtOH, 2-bottle choice) and (2) concomitant access, during which EtOH access continued with access to nicotine (0.03 mg/kg/infusion, i.v.) using a 2-lever choice procedure (active vs. inactive lever) in which the fixed ratio (FR) requirement was gradually increased to FR30. When stable co-use was obtained, rats were pretreated with varying doses of naltrexone, varenicline, or r-bPiDI, an α6β2* subtype-selective nicotinic acetylcholine receptor antagonist shown previously to reduce nicotine self-administration. Results While EtOH intake was initially suppressed in phase 2 (co-use), pharmacologically relevant intake for both substances was achieved by raising the "price" of nicotine to FR30. In phase 2, naltrexone decreased EtOH and water consumption but not nicotine intake; in contrast, naltrexone in phase 1 (EtOH only) did not significantly alter EtOH intake. Varenicline and r-bPiDI in phase 2 both decreased nicotine self-administration and inactive lever pressing, but neither altered EtOH or water consumption. Conclusions These results indicate that increasing the "price" of nicotine increases EtOH intake during co-use. Additionally, the efficacy of naltrexone, varenicline, and r-bPiDI was specific to either EtOH or nicotine, with no efficacy for co-use. Nevertheless, future studies on combining these treatments may reveal synergistic efficacy.

BACKGOUND:

Alcohol use disorder (AUD) is devastating and poorly treated, and innovative targets are actively sought for prevention and treatment. The orphan G protein-coupled receptor GPR88 is enriched in mesocorticolimbic pathways, and Gpr88 knockout mice show hyperactivity and risk-taking behavior, but a potential role for this receptor in drug abuse has not been examined.

METHODS:

We tested Gpr88 knockout mice for alcohol-drinking and -seeking behaviors. To gain system-level understanding of their alcoholendophenotype, we also analyzed whole-brain functional connectivity in naïve mice using resting-state functional magnetic resonance imaging.

RESULTS:

Gpr88 knockout mice showed increased voluntary alcohol drinking at both moderate and excessive levels, with intact alcoholsedation and metabolism. Mutant mice also showed increased operant responding and motivation for alcohol, while food and chocolate operant self-administration were unchanged. Alcohol place conditioning and alcohol-induced dopamine release in the nucleus accumbens were decreased, suggesting reduced alcohol reward in mutant mice that may partly explain enhanced alcohol drinking. Seed-based voxelwise functional connectivity analysis revealed significant remodeling of mesocorticolimbic centers, whose hallmark was predominant weakening of prefrontal cortex, ventral tegmental area, and amygdala connectional patterns. Also, effective connectivity from the ventral tegmental area to the nucleus accumbens and amygdala was reduced.

CONCLUSIONS:

Gpr88 deletion disrupts executive, reward, and emotional networks in a configuration that reduces alcohol reward and promotes alcohol seeking and drinking. The functional connectivity signature is reminiscent of alterations observed in individuals at risk for AUD. The Gpr88 gene, therefore, may represent a vulnerability/resilience factor for AUD, and a potential drug target for AUD treatment.

Individuals aged 12-20 years drink 11% of all alcohol consumed in the United States with more than 90% consumed in the form of bingedrinking. Early onset alcohol use is a strong predictor of future alcohol dependence. The study of the effects of excessive alcohol use on the human brain is hampered by limited information regarding the quantity and frequency of exposure to alcohol. Animal models can control for age at alcohol exposure onset and enable isolation of neural substrates of exposure to different patterns and quantities of ethanol (EtOH). As with humans, a frequently used binge exposure model is thought to produce dependence and affect predominantly corticolimbic brainregions. in vivo neuroimaging enables animals models to be examined longitudinally, allowing for each animal to serve as its own control. Accordingly, we conducted 3 magnetic resonance imaging (MRI) sessions (baseline, binge, recovery) to track structure throughout the brains of wild type Wistar rats to test the hypothesis that binge EtOH exposure affects specific brain regions in addition to corticolimbic circuitry. Voxel-based comparisons of 13 EtOH- vs. 12 water- exposed animals identified significant thalamic shrinkage and lateral ventricular enlargement as occurring with EtOH exposure, but recovering with a week of abstinence. By contrast, pretectal nuclei and superior and inferior colliculi shrank in response to binge EtOH treatment but did not recover with abstinence. These results identify brainstem structures that have been relatively underreported but are relevant for localizing neurocircuitry relevant to the dynamic course of alcoholism.

Long-term alcohol use can result in lasting changes in brain function, ultimately leading to alcohol dependence. These functional alterations arise from dysregulation of complex gene networks, and growing evidence implicates microRNAs as key regulators of these networks. We examined time- and brain region-dependent changes in microRNA expression after chronic intermittent ethanol (CIE) exposure in C57BL/6J mice. Animals were sacrificed at 0, 8, and 120h following the last exposure to four weekly cycles of CIE vapor and we measured microRNA expression in prefrontal cortex (PFC), nucleus accumbens (NAC), and amygdala (AMY). The number of detected (395–419) and differentially expressed (DE, 42–47) microRNAs was similar within each brain region. However, the DE microRNAs were distinct among brain regions and across time within each brain region. DE microRNAs were linked with their DE mRNA targets across each brain region. In all brain regions, the greatest number of DE mRNA targets occurred at the 0 or 8h time points and these changes were associated with microRNAs DE at 0 or 8h. Two separate approaches (discrete temporal association and hierarchical clustering) were combined with pathway analysis to further characterize the temporal relationships between DE microRNAs and their 120h DE targets. We focused on targets dysregulated at 120h as this time point represents a state of protracted withdrawal known to promote an increase in subsequent ethanol consumption. Discrete temporal association analysis identified networks with highly connected genes including ERK1/2 (mouse equivalent Mapk3, Mapk1), Bcl2 (in AMY networks) and Srf (in PFC networks). Similarly, the cluster-based analysis identified hub genes that include Bcl2 (in AMY networks) and Srf in PFC networks, demonstrating robust microRNA-mRNA network alterations in response to CIE exposure. In contrast, datasets utilizing targets from 0 and 8h microRNAs identified NF-kB-centered networks (in NAC and PFC), and Smad3-centered networks (in AMY). These results demonstrate that CIE exposure results in dynamic and complex temporal changes in microRNA-mRNA gene network structure.

While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.

Microglia are fundamentally important immune cells within the central nervous system (CNS) that respond to environmental challenges to maintain normal physiological processes. Alterations in steady-state cellular function and over-activation of microglia can facilitate the initiation and progression of neuropathological conditions such as Alzheimer's disease, Multiple Sclerosis, and Major Depressive Disorder. Alcohol consumption disrupts signaling pathways including both innate and adaptive immune responses that are necessary for CNS homeostasis. Coordinate expression of these genes is not ascertained from an admixture of CNS cell-types, underscoring the importance of examining isolated cellular populations to reveal systematic gene expression changes arising from mature microglia. Unbiased RNA-Seq profiling was used to identify gene expression changes in isolated prefrontal cortical microglia in response to recurring bouts of voluntary alcohol drinking behavior. The voluntary ethanol paradigm utilizes long-term consumption ethanol that results in escalated alcohol intake and altered cortical plasticity that is seen in humans. Gene coexpression analysis identified a coordinately regulated group of genes, unique to microglia, that collectively are associated with alcohol consumption. Genes within this group are involved in toll-like receptor signaling and transforming growth factor beta signaling. Network connectivity of this group identified Siglech as a putative hub gene and highlighted the potential importance of proteases in the microglial response to chronic ethanol. In conclusion, we identified a distinctive microglial gene expression signature for neuroimmune responses related to alcohol consumption that provides valuable insight into microglia-specific changes underlying the development of substance abuse, and possibly other CNS disorders.

Alcohol addiction leads to increased choice of alcohol over healthy rewards. We established an exclusive choice procedure in which ~15% of outbred rats chose alcohol over a high-value reward. These animals displayed addiction-like traits, including high motivation to obtain alcoholand pursuit of this drug despite adverse consequences. Expression of the γ-aminobutyric acid (GABA) transporter GAT-3 was selectively decreased within the amygdala of alcohol-choosing rats, whereas a knockdown of this transcript reversed choice preference of rats that originally chose a sweet solution over alcohol. GAT-3 expression was selectively decreased in the central amygdala of alcohol-dependent people compared to those who died of unrelated causes. Impaired GABA clearance within the amygdala contributes to alcohol addiction, appears to translate between species, and may offer targets for new pharmacotherapies for treating this disorder.

Excessive alcohol consumption in humans induces deficits in decision making and emotional processing, which indicates a dysfunction of the prefrontal cortex (PFC). The present study aimed to determine the impact of chronic intermittent ethanol (CIE) inhalation on mouse medial PFC pyramidal neurons. Data were collected 6–8 days into withdrawal from 7 weeks of CIE exposure, a time point when mice exhibit behavioral symptoms of withdrawal. We found that spine maturity in prelimbic (PL) layer 2/3 neurons was increased, while dendritic spines in PL layer 5 neurons or infralimbic (IL) neurons were not affected. Corroborating these morphological observations, CIE enhanced glutamatergic transmission in PL layer 2/3 pyramidal neurons, but not IL layer 2/3 neurons. Contrary to our predictions, these cellular alterations were associated with improved, rather than impaired, performance in reversal learning and strategy switching tasks in the Barnes maze at an earlier stage of chronic ethanol exposure (5–7 days withdrawal from 3 to 4 weeks of CIE), which could result from the anxiety-like behavior associated with ethanol withdrawal. Altogether, this study adds to a growing body of literature indicating that glutamatergic activity in the PFC is upregulated following chronic ethanol exposure, and identifies PL layer 2/3 pyramidal neurons as a sensitive target of synaptic remodeling. It also indicates that the Barnes maze is not suitable to detect deficits in cognitive flexibility in CIE-withdrawn mice.

Excessive alcohol consumption in humans induces deficits in decision making and emotional processing, which indicates a dysfunction of the prefrontal cortex (PFC). The present study aimed to determine the impact of chronic intermittent ethanol (CIE) inhalation on mouse medial PFC pyramidal neurons. Data were collected 6-8 days into withdrawal from 7 weeks of CIE exposure, a time point when mice exhibit behavioral symptoms of withdrawal. We found that spine maturity in prelimbic (PL) layer 2/3 neurons was increased, while dendritic spines in PL layer 5 neurons or infralimbic (IL) neurons were not affected. Corroborating these morphological observations, CIE enhanced glutamatergic transmission in PL layer 2/3 pyramidal neurons, but not IL layer 2/3 neurons. Contrary to our predictions, these cellular alterations were associated with improved, rather than impaired, performance in reversal learning and strategy switching tasks in the Barnes maze at an earlier stage of chronic ethanol exposure (5-7 days withdrawal from 3 to 4 weeks of CIE), which could result from the anxiety-like behavior associated with ethanol withdrawal. Altogether, this study adds to a growing body of literature indicating that glutamatergic activity in the PFC is upregulated following chronic ethanol exposure, and identifies PL layer 2/3 pyramidal neurons as a sensitive target of synaptic remodeling. It also indicates that the Barnes maze is not suitable to detect deficits in cognitive flexibility in CIE-withdrawn mice.

BACKGROUND:

Despite the high cost and widespread prevalence of alcohol use disorders, treatment options are limited, underscoring the need for new, effective medications. Previous results using protein kinase C epsilon (PKCε) knockout mice, RNA interference against PKCε, and peptide inhibitors of PKCε predict that small-molecule inhibitors of PKCε should reduce alcohol consumption in humans.

METHODS:

We designed a new class of PKCε inhibitors based on the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In vitro kinase and binding assays were used to identify the most potent compounds. Their effects on ethanol-stimulated synaptic transmission; ethanol, sucrose, and quinine consumption; ethanol-induced loss of righting; and ethanol clearance were studied in mice.

RESULTS:

We identified two compounds that inhibited PKCε with Ki <20 nM, showed selectivity for PKCε over other kinases, crossed the blood-brain barrier, achieved effective concentrations in mouse brain, prevented ethanol-stimulated gamma-aminobutyric acid release in the central amygdala, and reduced ethanol consumption when administered intraperitoneally at 40 mg/kg in wild-type but not in Prkce^-/- mice. One compound also reduced sucrose and saccharin consumption, while the other was selective for ethanol. Both transiently impaired locomotion through an off-target effect that did not interfere with their ability to reduce ethanol intake. One compound prolonged recovery from ethanol-induced loss of righting but this was also due to an off-target effect since it was present in Prkce^-/- mice. Neither altered ethanolclearance.

CONCLUSIONS:

These results identify lead compounds for development of PKCε inhibitors that reduce alcohol consumption.

Drug addiction is a worldwide societal problem and public health burden, and results from recreational drug use that develops into a complex brain disorder. The opioid system, one of the first discovered neuropeptide systems in the history of neuroscience, is central to addiction. Recently, opioid receptors have been propelled back on stage by the rising opioid epidemics, revolutions in G protein-coupled receptor research and fascinating developments in basic neuroscience. This Review discusses rapidly advancing research into the role of opioid receptors in addiction, and addresses the key questions of whether we can kill pain without addiction using mu-opioid-receptor-targeting opiates, how mu- and kappa-opioid receptors operate within the neurocircuitry of addiction and whether we can bridge human and animal opioid research in the field of drug abuse.

BACKGROUND:

Recently, the incidence of binge drinking by women has increased. Binge drinking is detrimental to women's health, yet the biological mechanisms that promote excessive drinking by women are not well understood. One method of assessing binge-like ethanol (EtOH) consumption in mice is the drinking in the dark (DID) test, in which mice drink sufficient EtOH to achieve intoxication. In this study, we directly compared male, female, and ovariectomized (OVX) mice for DID and tested whether 17β-estradiol (E2) contributes to DID. We also measured whether DID varies throughout the estrous cycle and whether repeated intermittent DID impacts the estrous cycle.

METHODS:

Male, female, and OVX C57BL/6J mice were tested for DID for 2 hours per day on days 1 to 3 and for 4 hours on day 4 using a single bottle containing 20% EtOH. To measure the effects of E2 on DID, OVX mice were treated with estradiol benzoate (EB) or vehicle daily starting 2 weeks prior to the drinking test and throughout the DID procedure. In a separate group of experiments, EtOH consumption and estrous cycle phase were measured in freely cycling mice that were drinking EtOH or water 5 days per week for 2 or 6 weeks.

RESULTS:

Female mice consumed more EtOH than male and OVX mice. Treatment with EB increased EtOH consumption by OVX mice compared with vehicle-treated controls. However, EtOH intake did not vary across the estrous cycle, nor did long-term DID alter the estrous cycle.

CONCLUSIONS:

These results demonstrate that ovarian hormones, specifically E2, contribute to increased EtOH consumption by female mice in the DID test. Although ovarian hormones contribute to this behavior, EtOH consumption is not affected by estrous cycle phase in freely cycling mice. This study provides a framework for understanding the factors that contribute to binge drinking in females.

Stress-related psychiatric disorders such as major depression are strongly associated with alcohol abuse and alcohol use disorder. Recently, many epidemiological and preclinical studies suggest that chronic stress prior to conception has cross-generational effects on the behaviorand physiological response to stress in subsequent generations. Thus, we hypothesized that chronic stress may also affect ethanol drinkingbehaviors in the next generation. In the first cohort of mice, we found that paternal preconception chronic variable stress significantly reduced both two-bottle choice and binge-like ethanol drinking selectively in male offspring. However, these results were not replicated in a second cohort that were tested under experimental conditions that were nearly identical, except for one notable difference. Cohort 1 offspring were derived from in-house C57BL/6J sires that were born in the animal vivarium at the University of Pittsburgh whereas cohort 2 offspring were derived from C57BL/6J sires shipped directly from the vendor. Therefore, a third cohort that included both in-house and vendor born sires was analyzed. Consistent with the first two cohorts, we observed a significant interaction between chronic stress and sire-source with only stressed sires that were born in-house able to impart reduced ethanol drinking behaviors to male offspring. Overall, these results demonstrate that paternal preconception stress can impact ethanol drinking behavior in males of the next generation. These studies provide additional support for a recently recognized role of the paternal preconception environment in shaping ethanol drinking behavior.

Impairment in glutamate neurotransmission mediates the development of dependence upon nicotine (NIC) and ethanol (EtOH). Previous work indicates that continuous access to EtOH or phasic exposure to NIC reduces expression of the glutamate transporter-1 (GLT-1) and cystine/glutamate antiporter (xCT) but not the glutamate/aspartate transporter (GLAST). Additionally, metabotropic glutamate receptors (mGluRs) expression was affected following exposure to EtOH or NIC. However, little is known about the effects of EtOH and NIC co-consumption on GLT-1, xCT, GLAST, and mGluR1 expression. In this study, peri-adolescent female alcohol preferring (P) rats were given binge-like access to water, sucrose (SUC), SUC-NIC, EtOH, or EtOH-NIC for four weeks. The present study determined the effects of these reinforcers on GLT-1, xCT, GLAST, and mGluR1 expression in the nucleus accumbens (NAc), hippocampus (HIP) and prefrontal cortex (PFC). GLT-1 and xCT expression were decreased in the NAc following both SUC-NIC and EtOH-NIC. In addition, only xCT expression was downregulated in the HIP in both of these latter groups. Also, glutathione peroxidase (GPx) activity in the HIP was reduced following SUC, SUC-NIC, EtOH, and EtOH-NIC consumption. Similar to previous work, GLAST expression was not altered in any brain region by any of the reinforcers. However, mGluR1 expression was increased in the NAc in the SUC-NIC, EtOH, and EtOH-NIC groups. These results indicate that peri-adolescent binge-like drinking of EtOH or SUC with or without NIC may exert differential effects on astroglial glutamate transporters and receptors. Our data further parallel some of the previous findings observed in adult rats.