Hauser SR, Knight CP, Truitt WA, Waeiss RA, Holt IS, Carvajal GB, Bell RL, Rodd ZA. Adolescent intermittent ethanol increases the sensitivity to the reinforcing properties of ethanol and the expression of select cholinergic and dopaminergic genes within the posterior ventral tegmental area. Alcoholism: Clinical and Experimental Research [Internet]. 2019 :43:1937-1948. Publisher's VersionAbstract


Although not legally allowed to consume alcohol, adolescents account for 11% of all alcohol use in the United States and approximately 90% of adolescent intake is in the form of an alcohol binge. The adolescent intermittent ethanol (AIE) model developed by the NADIA consortium produces binge‐like EtOH exposure episodes. The current experiment examined the effects of AIE on the reinforcing properties of EtOH and genetic expression of cholinergic and dopaminergic factors within the posterior ventral tegmental area (pVTA) in Wistar male and female rats and in male alcohol‐preferring (P) rats.


Rats were exposed to the AIE or water during adolescence, and all testing occurred during adulthood. Wistar control and AIE rats were randomly assigned to groups that self‐administered 0 to 200 mg% EtOH. Male P rats self‐administered 0 to 100 mg%.


The data indicated that exposure to AIE in both Wistar male and female rats (and male P rats) resulted in a significant leftward shift in dose–response curve for EtOH self‐administration into the pVTA. TaqMan array indicated that AIE exposure had divergent effects on the expression of nicotinic receptors (increased a7, reduction in a4 and a5). There were also sex‐specific effects of AIE on gene expression; male only reduction in D3 receptors.


Binge‐like EtOH exposure during adolescence enhances the sensitivity to the reinforcing properties of EtOH during adulthood which could be part of biological sequelae that are the basis for the deleterious effects of adolescent alcohol consumption on the rate of alcoholism during adulthood.

Hauser SR, Deehan GAJ, Knight CP, Waeiss RA, Truitt WA, Johnson PL, Bell RL, McBride WJ, Rodd ZA. Conditioned stimuli affect ethanol-seeking by female alcohol-preferring (P) rats: the role of repeated-deprivations, cue-pretreatment, and cue-temporal intervals. Psychopharmacology [Internet]. 2019 :236:2835-2846. Publisher's VersionAbstract


Evidence indicates that drug-paired stimuli can evoke drug-craving leading to drug-seeking and repeated relapse periods can influence drug-seeking behaviors.


The present study examined (1) the effect of an interaction between repeated deprivation cycles and excitatory conditioning stimuli (CS+) on ethanol (EtOH)-seeking; (2) the effects of EtOH-paired cue-exposure in a non-drug-paired environment on subsequent conditioning in a drug-paired environment; and (3) the temporal effects of conditioned cues on subsequent EtOH-seeking.


Adult female alcohol-preferring (P) rats were exposed to three conditioned odor cues; CS+ associated with EtOH self-administration, CS- associated with the absence of EtOH (extinction training), and a neutral stimulus (CS0) presented in a neutral non-drug-paired environment. The rats underwent four deprivation cycles or were non-deprived, following extinction they were maintained in a home cage for an EtOH-free period, and then exposed to no cue, CS+, CS-, or CS0 to assess the effect of the conditioned cues on EtOH-seeking behavior.


Repeated deprivations enhanced and prolonged the duration of CS+ effects on EtOH-seeking. Presentation of the CS- in a non-drug-paired environment blocked the ability of a CS+ to enhance EtOH-seeking in a drug-paired environment. Presentation of the CS+ or CS- in a non-drug-paired environment 2 or 4 h earlier significantly altered EtOH-seeking.


Results indicated an interaction between repeated deprivation cycles and CS+ resulted in a potentiation of CS+ evoked EtOH-seeking. In addition, a CS- may have therapeutic potential by providing prophylactic protection against relapse behavior in the presence of cues in the drug-using environment.

Erickson EK, Blednov YA, Harris RA, Mayfield RD. Glial gene networks associated with alcohol dependence. Scientific Reports [Internet]. 2019 :9:10949. Publisher's VersionAbstract
Chronic alcohol abuse alters the molecular structure and function of brain cells. Recent work suggests adaptations made by glial cells, such as astrocytes and microglia, regulate physiological and behavioral changes associated with addiction. Defining how alcohol dependence alters the transcriptome of different cell types is critical for developing the mechanistic hypotheses necessary for a nuanced understanding of cellular signaling in the alcohol-dependent brain. We performed RNA-sequencing on total homogenate and glial cell populations isolated from mouse prefrontal cortex (PFC) following chronic intermittent ethanol vapor exposure (CIE). Compared with total homogenate, we observed unique and robust gene expression changes in astrocytes and microglia in response to CIE. Gene co-expression network analysis revealed biological pathways and hub genes associated with CIE in astrocytes and microglia that may regulate alcohol-dependent phenotypes. Astrocyte identity and synaptic calcium signaling genes were enriched in alcohol-associated astrocyte networks, while TGF-β signaling and inflammatory response genes were disrupted by CIE treatment in microglia gene networks. Genes related to innate immune signaling, specifically interferon pathways, were consistently up-regulated across CIE-exposed astrocytes, microglia, and total homogenate PFC tissue. This study illuminates the cell-specific effects of chronic alcohol exposure and provides novel molecular targets for studying alcohol dependence.
Chung T, Harris RA. Cannabis and alcohol: from basic science to public policy. Alcoholism: Clinical and Experimental Research [Internet]. 2019 :43:1829-1833. Publisher's VersionAbstract
The emergence of state-level approval of cannabis for both medical and recreational use is likely to increase the already prevalent co-use of alcohol and cannabis (Yurasek et al., 2017) and raise many important health and social concerns (National Academies of Sciences, 2017). Cannabis research has lagged behind that of alcohol research, but important studies are emerging on the interactions between alcohol and cannabinoids. In this Virtual Issue, Cannabis and Alcohol: From Basic Science to Public Policy, we present 9 leading-edge research publications spanning preclinical and epidemiological studies, as well as a critical review of the potential therapeutic use of cannabidiol (CBD) in the treatment of alcohol use disorder (AUD) (Turna et al., 2019), which recently appeared in Alcoholism: Clinical and Experimental Research. The Virtual Issue addresses the potential risks and benefits of alcohol and cannabis co-use, which may depend on the particular subgroup of individuals, and whether these drugs are used simultaneously (i.e., drug effects overlap) (Pakula et al., 2009) or concurrently (i.e., drug effects do not overlap in time) (Subbaraman et al., 2019). Simultaneous drug use may be perceived as a means to complement or enhance the effects of each substance (Patrick et al., 2018), despite some individuals reporting negative effects (Lee et al., 2017). The positive perception of complementary drug effects is concerning given the greater health risks associated with simultaneous use (Volkow et al., 2014; Yurasek et al., 2017). Another pattern of use has also developed that substitutes cannabis for alcohol use, particularly in individuals who are making efforts to reduce alcohol intake (Subbaraman, 2016). As summarized in the sections below, this Virtual Issue provides a current assessment of cannabis–alcohol interactions and shows patterns of drug use and risk profiles that may impact the prevalence of co-use and dependence.
Chen WY, Zhang H, Gatta E, Glover EJ, Pandey SC, Lasek AW. The histone deacetylase inhibitorsuberoylanilide hydroxamic acid (SAHA) alleviates depression-like behavior and normalizes epigeneticchanges in the hippocampus during ethanol withdrawal. Alcohol [Internet]. 2019 :78:79-87. Publisher's VersionAbstract
Withdrawal from chronic alcohol drinking can cause depression, leading to an inability to function in daily life and an increased risk for relapse to harmful drinking. Understanding the causes of alcohol withdrawal-related depression may lead to new therapeutic targets for treatment. Epigenetic factors have recently emerged as important contributors to both depression and alcohol use disorder (AUD). Specifically, acetylation of the N-terminal tails of histone proteins that package DNA into nucleosomes is altered in stress-induced models of depression and during alcohol withdrawal. The goal of this study was to examine depression-like behavior during alcohol withdrawal and associated changes in histone acetylation and expression of histone deacetylase 2 (HDAC2) in the hippocampus, a brain region critical for mood regulation and depression. Male Sprague-Dawley rats were treated with the Lieber-DeCarli ethanol liquid diet for 15 days and then underwent withdrawal. Rats were treated with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), during withdrawal and were tested for depression-like behavior. In a separate group of rats, the hippocampus was analyzed for mRNA and protein expression of HDAC2 and levels of histone H3 lysine 9 acetylation (H3K9ac) during chronic ethanol exposure and withdrawal. Rats undergoing ethanol withdrawal exhibited depression-like behavior and had increased HDAC2 and decreased H3K9ac levels in specific structures of the hippocampus. Treatment with SAHA during withdrawal ameliorated depression-like behavior and normalized changes in hippocampal HDAC2 and H3K9ac levels. These results demonstrate that ethanol withdrawal causes an altered epigenetic state in the hippocampus. Treatment with an HDAC inhibitor can correct this state and alleviate depression-like symptoms developed during withdrawal. Targeting histone acetylation may be a novel strategy to reduce ethanol withdrawal-induced depression.
Bickel WK, Crabbe JC, Sher KJ. What Is addiction? How can animal and human research be used to advance research, diagnosis, and treatment of alcohol and other substance use disorders?. Alcoholism: Clinical and Experimental Research [Internet]. 2019;43 :6-21. Publisher's VersionAbstract

The current article highlights key issues in defining, studying, and treating addiction, a concept related to but distinct from substance use disorders. The discussion is based upon a roundtable discussion at the 2017 annual meeting of the Research Society on Alcoholism where Warren K. Bickel and John C. Crabbe were charged with answering a range of questions posed by Kenneth J. Sher. All the presenters highlighted a number of central concerns for those interested in assessing and treating addiction as well as those seeking to conduct basic preclinical research that is amenable to meaningful translation to the human condition. In addition, the discussion illustrated both the power and limitations of using any single theory to explain multiple phenomena subsumed under the rubric of addiction. Among the major issues examined were the important differences between traditional diagnostic approaches and current concepts of addiction, the difficulty of modeling key aspects of human addiction in nonhuman animals, key aspects of addiction that have, to date, received little empirical attention, and the importance of thinking of recovery as a phenomenon that possibly involves processes distinct from those undergirding the development and maintenance of addiction.

de Guglielmo G, Kallupi M, Pomrenze MB, Crawford E, Simpson S, Schweitzer P, Koob GF, Messing RO, George O. Inactivation of a CRF-dependent amygdalofugal pathway reverses addiction-like behaviors in alcohol-dependent rats. Nature Communications [Internet]. 2019;2019 :1238. Publisher's VersionAbstract
The activation of a neuronal ensemble in the central nucleus of the amygdala (CeA) during alcohol withdrawal has been hypothesized to induce high levels of alcohol drinking in dependent rats. In the present study we describe that the CeA neuronal ensemble that is activated by withdrawal from chronic alcohol exposure contains ~80% corticotropin-releasing factor (CRF) neurons and that the optogenetic inactivation of these CeA CRF+ neurons prevents recruitment of the neuronal ensemble, decreases the escalation of alcohol drinking, and decreases the intensity of somatic signs of withdrawal. Optogenetic dissection of the downstream neuronal pathways demonstrates that the reversal of addiction-likebehaviors is observed after the inhibition of CeA CRF projections to the bed nucleus of the stria terminalis (BNST) and that inhibition of the CRFCeA-BNST pathway is mediated by inhibition of the CRF-CRF1 system and inhibition of BNST cell firing. These results suggest that the CRFCeA-BNST pathway could be targeted for the treatment of excessive drinking in alcohol use disorder.
Ferguson LB, Zhang L, Kircher D, Wang S, Mayfield RD, Crabbe JC, Morrisett RA, Harris RA, Ponomarev I. Dissecting brain networks underlying alcohol binge drinking using a systems genomics approach. Molecular Neurobiology [Internet]. 2019;56 :2791-2810. Publisher's VersionAbstract
Alcohol use disorder (AUD) is a complex psychiatric disorder with strong genetic and environmental risk factors. We studied the molecular perturbations underlying risky drinking behavior by measuring transcriptome changes across the neurocircuitry of addiction in a genetic mouse model of binge drinking. Sixteen generations of selective breeding for high blood alcohol levels after a binge drinking session produced global changes in brain gene expression in alcohol-naïve High Drinking in the Dark (HDID-1) mice. Using gene expression profiles to generate circuit-level hypotheses, we developed a systems approach that integrated regulation of gene coexpression networks across multiple brain regions, neuron-specific transcriptional signatures, and knowledgebase analytics. Whole-cell, voltage-clamp recordings from nucleus accumbens shell neurons projecting to the ventral tegmental area showed differential ethanol-induced plasticity in HDID-1 and control mice and provided support for one of the hypotheses. There were similarities in gene networks between HDID-1 mouse brains and postmortem brains of human alcoholics, suggesting that some gene expression patterns associated with high alcohol consumption are conserved across species. This study demonstrated the value of gene networks for data integration across biological modalities and species to study mechanisms of disease.
Kircher DM, Aziz HC, Mangieri RA, Morrisett RA. Ethanol experience enhances glutamatergic ventral hippocampal inputs to D1 receptor-expressing medium spiny neurons in the nucleus accumbens shell. Journal of Neuroscience [Internet]. 2019;39 :2459-2469. Publisher's VersionAbstract

A growing number of studies implicate alterations in glutamatergic signaling within the reward circuitry of the brain during alcohol abuse and dependence. A key integrator of glutamatergic signaling in the reward circuit is the nucleus accumbens, more specifically, the dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) within this region, which have been implicated in the formation of dependence to many drugs of abuse including alcohol. D1-MSNs receive glutamatergicinput from several brain regions; however, it is not currently known how individual inputs onto D1-MSNs are altered by alcohol experience. Here, we investigate input-specific adaptations in glutamatergic transmission in response to varying levels of alcohol experience. Virally mediated expression of Channelrhodopsin in ventral hippocampal (vHipp) glutamate neurons of male mice allowed for selective activation of vHipp to D1-MSN synapses. Therefore, we were able to compare synaptic adaptations in response to low and high alcohol experience in vitro and in vivo Alcohol experience enhanced glutamatergic activity and abolished LTD at vHipp to D1-MSN synapses. Following chronic alcohol experience, GluA2-lacking AMPARs, which are Ca permeable, were inserted into vHipp to D1-MSN synapses. These findings support the reversal of alcohol-induced insertion of Ca-permeable AMPARs and the enhancement of glutamatergic activity at vHipp to D1-MSNs as potential targets for intervention during early exposure to alcohol. SIGNIFICANCE STATEMENT Given the roles of the nucleus accumbens (NAc) in integrating cortical and allocortical information and in reward learning, it is vital to understand how inputs to this region are altered by drugs of abuse such as alcohol. The strength of excitatory inputs from the ventral hippocampus (vHipp) to the NAc has been positively associated with reward-related behaviors, but it is unclear whether or how ethanol affects these inputs. Here we show that vHipp-NAc synapses indeed are altered by ethanolexposure, with vHipp glutamatergic input to the NAc being enhanced following chronic ethanol experience. This work provides insight into ethanol-induced alterations of vHipp-NAc synapses and suggests that, similarly to drugs such as cocaine, the strengthening of these synapses promotes reward behavior.

Mulligan MK, Abreo T, Neuner SM, Parks C, Watkins CE, Houseal MT, Shapaker TM, Hook M, Tan H, Wang X, et al. Identification of a functional non-coding variant in the GABA (A) receptor α2 subunit of the C57BL/6J mouse reference genome: major implications for neuroscience research. Frontiers in Genetics [Internet]. 2019;10 :188. Publisher's VersionAbstract
GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR-Cas9-mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.
Zahr NM, Pohl KM, Saranathan M, Sullivan EV, Pfefferbaum A. Hippocampal subfield CA2+3 exhibits accelerated aging in alcohol use disorder: a preliminary study. Neuroimage: Clinical. 2019;22 :101764.Abstract
The profile of brain structural dysmorphology of individuals with Alcohol Use Disorders (AUD) involves disruption of the limbic system. In vivo imaging studies report hippocampal volume loss in AUD relative to controls, but only recently has it been possible to articulate different regions of this complex structure. Volumetric analysis of hippocampal regions rather than total hippocampal volume may augment differentiation of disease processes. For example, damage to hippocampal subfield cornu ammonis 1 (CA1) is often reported in Alzheimer's disease (AD), whereas deficits in CA4/dentate gyrus are described in response to stress and trauma. Two previous studies explored the effects of chronic alcohol use on hippocampal subfields: one reported smaller volume of the CA2+3 in alcohol-dependent subjects relative to controls, associated with years of alcohol consumption; the other, smaller volumes of presubiculum, subiculum, and fimbria in alcohol-dependent relative to control men. The current study, conducted in 24 adults with DSM5-diagnosed AUD (7 women, 53.7 ± 8.8) and 20 controls (7 women, 54.1 ± 9.3), is the first to use FreeSurfer 6.0, which provides state-of-the art hippocampal parcellation, to explore the sensitivity of hippocampal sufields to alcoholism. T1- and T2- images were collected on a GE MR750 system with a 32-channel Nova head coil. FreeSurfer 6.0 hippocampal subfield analysis produced 12 subfields: parasubiculum; presubiculum; subiculum; CA1; CA2+3; CA4; GC-ML-DG (Granule Cell (GC) and Molecular Layer (ML) of the Dentate Gyrus (DG)); molecular layer; hippocampus-amygdala-transition-area (HATA); fimbria; hippocampal tail; hippocampal fissure; and whole volume for left and right hippocampi. A comprehensive battery of neuropsychological tests comprising attention, memory and learning, visuospatial abilities, and executive functions was administered. Multiple regression analyses of raw volumetric data for each subfields by group, age, sex, hemisphere, and supratentorial volume (svol) showed significant effects of svol (p < .04) on nearly all structures (excluding tail and fissure). Volumes corrected for svol showed effects of age (fimbria, fissure) and group (subiculum, CA1, CA4, GC-ML-DG, HATA, fimbria); CA2+3showed a diagnosis-by-age interaction indicating older AUD individuals had a smaller volume than would be expected for their age. There were no selective relations between hippocampalsubfields and performance on neuropsychological tests, likely due to lack of statistical power. The current results concur with the previous study identifying CA2+3 as sensitive to alcoholism, extend them by identifying an alcoholism-age interaction, and suggest an imaging phenotype distinguishing AUD from AD and stress/trauma.
You C, Savarese A, Vandegrift BJ, He D, Pandey SC, Lasek AW, Brodie MS. Ethanol acts on KCNK13 potassium channels in the ventral tegmental area to increase firing rate and modulate binge-like drinking. Neuropharmacology. 2019;144 :29-36.Abstract
Alcohol excitation of the ventral tegmental area (VTA) is important in neurobiological processes related to the development of alcoholism. The ionotropic receptors on VTA neurons that mediate ethanol-induced excitation have not been identified. Quinidine blocks ethanol excitation of VTA neurons, and blockade of two-pore potassium channels is among the actions of quinidine. Therefore two-pore potassium channels in the VTA may be potential targets for the action of ethanol. Here, we explored whether ethanol activation of VTA neurons is mediated by the two-pore potassium channel KCNK13. Extracellular recordings of the response of VTA neurons to ethanol were performed in combination with knockdown of Kcnk13 using a short hairpin RNA (shRNA) in C57BL/6 J mice. Real-time PCR and immunohistochemistry were used to examine expression of this channel in the VTA. Finally, the role of KCNK13 in binge-like drinking was examined in the drinking in the dark test after knockdown of the channel. Kcnk13 expression in the VTA was increased by acute ethanol exposure. Ethanol-induced excitation of VTA neurons was selectively reduced by shRNA targeting Kcnk13. Importantly, knockdown of Kcnk13 in the VTA resulted in increased alcohol drinking. These results are consistent with the idea that ethanol stimulates VTA neurons at least in part by inhibiting KCNK13, a specific two-pore potassium channel, and that KCNK13 can control both VTA neuronal activity and binge drinking. KCNK13 is a novel alcohol-sensitive molecular target and may be amenable to the development of pharmacotherapies for alcoholism treatment.
Wolfe SA, Farris SP, Mayfield JE, Heaney CF, Erickson EK, Harris RA, Mayfield RD, Raab-Graham KF. Ethanol and a rapid-acting antidepressant produce overlapping changes in exon expression in the synaptic transcriptome. Neuropharmacology. 2019;146 :289-299.Abstract
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.
Warden AS, Azzam M, Da Costa A, Mason S, Blednov YA, Messing RO, Mayfield RD, Harris RA. Toll-like receptor 3 dynamics in female C57BL/6J mice: regulation of alcohol intake. Brain Behavior and Immunity. 2019;77 :66-76.Abstract
Although there are sex differences in the effects of alcohol on immune responses, it is unclear if sex differences in immune response can influence drinking behavior. Activation of toll-like receptor 3 (TLR3) by polyinosinic:polycytidylic acid (poly(I:C)) produced a rapid proinflammatory response in males that increased alcohol intake over time (Warden et al., 2019). Poly(I:C) produced a delayed and prolonged innate immune response in females. We hypothesized that the timecourse of innate immune activation could regulate drinking behavior in females. Therefore, we chose to test the effect of two time points in the innate immune activation timecourse on every-other-day two-bottle-choice drinking: (1) peak activation; (2) descending limb of activation. Poly(I:C) reduced ethanol consumption when alcohol access occurred during peak activation. Poly(I:C) did not change ethanol consumption when alcohol access occurred on the descending limb of activation. Decreased levels of MyD88-dependent pathway correlated with decreased alcohol intake and increased levels of TRIF-dependent pathway correlated with increased alcohol intake in females. To validate the effects of poly(I:C) were mediated through MyD88, we tested female mice lacking Myd88. Poly(I:C) did not change alcohol intake in Myd88 knockouts, indicating that poly(I:C)-induced changes in alcohol intake are dependent on MyD88 in females. We next determined if the innate immune timecourse also regulated drinking behavior in males. Poly(I:C) reduced ethanol consumption in males when alcohol was presented at peak activation. Therefore, the timecourse of innate immune activation regulates drinking behavior and sex-specific dynamics of innate immune response must be considered when designing therapeutics to treat excessive drinking
Warden AS, Azzam M, Da Costa A, Mason S, Blednov YA, Messing RO, Mayfield RD, Harris RA. Toll-like receptor 3 activation increases voluntary alcohol intake in C57BL/6J male mice. Brain Behavior and Immunity. 2019;77 :55-65.Abstract
Many genes differentially expressed in brain tissue from human alcoholics and animals that have consumed large amounts of alcohol are components of the innate immune toll-like receptor (TLR) pathway. TLRs initiate inflammatory responses via two branches: (1) MyD88-dependent or (2) TRIF-dependent. All TLRs signal through MyD88 except TLR3. Prior work demonstrated a direct role for MyD88-dependent signaling in regulation of alcohol consumption. However, the role of TLR3 as a potential regulator of excessive alcohol drinking has not previously been investigated. To test the possibility TLR3 activation regulates alcohol consumption, we injected mice with the TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) and tested alcohol consumption in an every-other-day two-bottle choice test. Poly(I:C) produced a persistent increase in alcohol intake that developed over several days. Repeated poly(I:C) and ethanol exposure altered innate immune transcript abundance; increased levels of TRIF-dependent pathway components correlated with increased alcohol consumption. Administration of poly(I:C) before exposure to alcohol did not alter alcohol intake, suggesting that poly(I:C) and ethanol must be present together to change drinking behavior. To determine which branch of TLR signaling mediates poly(I:C)-induced changes in drinking behavior, we tested either mice lacking MyD88 or mice administered a TLR3/dsRNA complex inhibitor. MyD88 null mutants showed poly(I:C)-induced increases in alcohol intake. In contrast, mice pretreated with a TLR3/dsRNA complex inhibitor reduced their alcohol intake, suggesting poly(I:C)-induced escalations in alcohol intake are, at least partially, dependent on TLR3. Together, these results strongly suggest that TLR3-dependent signaling drives excessive alcohol drinking behavior.
Patel RR, Khom S, Steinman MQ, FP V, Kiosses WB, Hedges DM, Vlkolinsky R, Nadav T, Polis I, Bajo M, et al. IL-1β expression is increased and regulates GABA transmission following chronic ethanol in mouse central amygdala. Brain, Behavior and Immunity. 2019;75 :208-219.Abstract
The interleukin-1 system (IL-1) is a prominent pro-inflammatory pathway responsible for the initiation and regulation of immune responses. Human genetic and preclinical studies suggest a critical role for IL-1β signaling in ethanol drinking and dependence, but little is known about the effects of chronic ethanol on the IL-1 system in addiction-related brain regions such as the central amygdala (CeA). In this study, we generated naïve, non-dependent (Non-Dep) and dependent (Dep) male mice using a paradigm of chronic-intermittent ethanol vapor exposure interspersed with two-bottle choice to examine 1) the expression of IL-1β, 2) the role of the IL-1 system on GABAergic transmission, and 3) the potential interaction with the acute effects of ethanol in the CeA. Immunohistochemistry with confocal microscopy was used to assess expression of IL-1β in microglia and neurons in the CeA, and whole-cell patch clamp recordings were obtained from CeA neurons to measure the effects of IL-1β (50 ng/ml) or the endogenous IL-1 receptor antagonist (IL-1ra; 100 ng/ml) on action potential-dependent spontaneous inhibitory postsynaptic currents (sIPSCs). Overall, we found that IL-1β expression is significantly increased in microglia and neurons of Dep compared to Non-Dep and naïve mice, IL-1β and IL-1ra bi-directionally modulate GABA transmission through both pre- and postsynaptic mechanisms in all three groups, and IL-1β and IL-1ra do not alter the facilitation of GABA release induced by acute ethanol. These data suggest that while ethanol dependence induces a neuroimmune response in the CeA, as indicated by increased IL-1β expression, this does not significantly alter the neuromodulatory role of IL-1β on synaptic transmission.
Mittal N, Thakore N, Bell RL, Maddox WT, Schallert T, Duvauchelle CL. Sex-specific ultrasonic vocalization patterns and alcohol consumption in high alcohol-drinking (HAD-1) rats. Physiology and Behavior. 2019;203 :81-90.Abstract
Ultrasonic vocalizations (USVs) have been established as an animal model of emotional status and are often utilized in drug abuse studies as motivational and emotional indices. Further USV functionality has been demonstrated in our recent work showing accurate identification of selectively-bred high versus low alcohol-consuming male rats ascertained exclusively from 22 to 28kHz and 50-55kHz FM USV acoustic parameters. With the hypothesis that alcohol-sensitive sex differences could be revealed through USV acoustic parameters, the present study examined USVs and alcohol consumption in male and female selectively bred high-alcohol drinking (HAD-1) rats. For the current study, we examined USV data collected during a 12-week experiment in male and female HAD-1 rats. Experimental phases included Baseline (2weeks), 4-h EtOH Access (4weeks), 24-h EtOH Access (4weeks) and Abstinence (2weeks). Findings showed that both male and female HAD-1 rats spontaneously emitted a large number of 22-28kHz and 50-55kHz FM USVs and that females drank significantly more alcohol compared to males over the entire course of the experiment. Analyses of USV acoustic characteristics (i.e. mean frequency, duration, bandwidth and power) revealed distinct sex-specific phenotypes in both 50-55kHz FM and 22-28kHz USV transmission that were modulated by ethanol exposure. Moreover, by using a linear combination of these acoustic characteristics, we were able to develop binomial logistic regression models able to discriminate between male and female HAD-1 rats with high accuracy. Together these results highlight unique emotional phenotypes in male and female HAD-1 rats that are differentially modulated by alcohol experience.
Kapoor M, Wang JC, Farris SP, Liu Y, McClintick J, Gupta I, Meyers JL, Bertelsen S, Chao M, Nurnberger J, et al. Analysis of whole genome-transcriptomic organization in brain to identify genes associated with alcoholism. Translational Psychiatry. 2019;9 :89.Abstract
Alcohol exposure triggers changes in gene expression and biological pathways in human brain. We explored alterations in gene expression in the Pre-Frontal Cortex (PFC) of 65 alcoholics and 73 controls of European descent, and identified 129 genes that showed altered expression (FDR < 0.05) in subjects with alcohol dependence. Differentially expressed genes were enriched for pathways related to interferon signaling and Growth Arrest and DNA Damage-inducible 45 (GADD45) signaling. A coexpression module (thistle2) identified by weighted gene co-expression network analysis (WGCNA) was significantly correlated with alcohol dependence, alcohol consumption, and AUDIT scores. Genes in the thistle2 module were enriched with genes related to calcium signaling pathways and showed significant downregulation of these pathways, as well as enrichment for biological processes related to nicotine response and opioid signaling. A second module (brown4) showed significant upregulation of pathways related to immune signaling. Expression quantitative trait loci (eQTLs) for genes in the brown4 module were also enriched for genetic associations with alcohol dependence and alcohol consumption in large genome-wide studies included in the Psychiatric Genetic Consortium and the UK Biobank's alcohol consumption dataset. By leveraging multi-omics data, this transcriptome analysis has identified genes and biological pathways that could provide insight for identifying therapeutic targets for alcohol dependence.
Jimenez VA, Herman MA, Cuzon Carlson VC, Walter NA, Grant KA, Roberto M. Synaptic adaptations in the central amygdala and hypothalamic paraventricular nucleus associated with protracted ethanol abstinence in male rhesus monkeys. Neuropsychopharmacology. 2019;44 :982-993.Abstract

Alcohol use disorder is a significant global burden. Stress has been identified as an etiological factor in the initiation and continuation of ethanol consumption. Understanding adaptations within stress circuitry is an important step toward novel treatment strategies. The effects of protracted abstinence following long-term ethanol self-administration on the central nucleus of the amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) were evaluated in male rhesus monkeys. Using whole-cell patch-clamp electrophysiology, inhibitory GABAergic transmission in the CeA and excitatory glutamatergic transmission in the PVN were measured. CeA neurons from abstinent drinkers displayed an elevated baseline spontaneous inhibitory postsynaptic current (sIPSC) frequency compared with controls, indicating increased presynaptic GABA release. Application of acute ethanol significantly increased the frequency of sIPSCs in controls, but not in abstinent drinkers, suggesting a tolerance to ethanol-enhanced GABA release in abstinent rhesus monkeys with a history of chronic ethanol self-administration and repeated abstinence. In the PVN, the frequency of spontaneous excitatory postsynaptic currents (sEPSC) was elevated in abstinent drinkers compared with controls, indicating increased presynaptic glutamate release. Notably, acute ethanol decreased presynaptic glutamate release onto parvocellular PVN neurons in both controls and abstinent drinkers, suggesting a lack of tolerance to acute ethanol among PVN neurons. These results are the first to demonstrate distinct synaptic adaptations and ethanol sensitivity in both the extrahypothalamic and hypothalamic stress circuits in abstinent rhesus males. Importantly, our findings describe adaptations in stress circuitry present in the brain at a state during abstinence, just prior to relapse to ethanol drinking.


Erickson EK, Grantham EK, Warden AS, Harris RA. Neuroimmune signaling in alcohol use disorder. Pharmacology Biochemistry and Behavior. 2019;177 :34-60.Abstract
Alcohol use disorder (AUD) is a widespread disease with limited treatment options. Targeting the neuroimmune system is a new avenue for developing or repurposing effective pharmacotherapies. Alcohol modulates innate immune signaling in different cell types in the brain by altering gene expression and the molecular pathways that regulate neuroinflammation. Chronic alcohol abuse may cause an imbalance in neuroimmune function, resulting in prolonged perturbations in brain function. Likewise, manipulating the neuroimmune system may change alcohol-related behaviors. Psychiatric disorders that are comorbid with AUD, such as post-traumatic stress disorder, major depressive disorder, and other substance use disorders, may also have underlying neuroimmune mechanisms; current evidence suggests that convergent immune pathways may be involved in AUD and in these comorbid disorders. In this review, we provide an overview of major neuroimmune cell-types and pathways involved in mediating alcohol behaviors, discuss potential mechanisms of alcohol-induced neuroimmune activation, and present recent clinical evidence for candidate immune-related drugs to treat AUD.