In Press
Okhuarobo A, Kreifeldt M, Gandhi PJ, Lopez C, Martinez B, Fleck K, Bajo M, Bhattacharyya P, Dopico AM, Roberto M, Roberts AJ, Homanics GE, and Contet C. “Ethanol's interaction with BK channel α subunitresidue K361 does not mediate behavioral responses to alcohol in mice..” Mol Psychiatry. Publisher's Version Abstract
Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol’s direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary β subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.
Aguilar J, De Carvalho LM, Chen H, Condon R, Lasek AW, and Pradhan AA. “Histone deacetylase inhibitor decreases hyperalgesia in a mouse model of alcohol withdrawal-induced hyperalgesia.” Alcohol, clinical & experimental research. Publisher's Version Abstract

Background: Alcohol withdrawal-induced hyperalgesia (AWH) is characterized as an increased pain sensitivity observed after cessation of chronic alcohol use. Alcohol withdrawal-induced hyperalgesia can contribute to the negative affective state associated with abstinence and can increase susceptibility to relapse. We aimed to characterize pain sensitivity in mice during withdrawal from two different models of alcohol exposure: chronic drinking in the dark (DID) and the Lieber-DeCarli liquid diet. We also investigated whether treatment with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), could ameliorate AWH in mice treated with the Lieber-DeCarli diet.

Methods: Male and female C57BL/6J mice were used for these studies. In the DID model, mice received bottles of 20% ethanol or water during the dark cycle for 4 h per day on four consecutive days per week for 6 weeks. Peripheral mechanical sensitivity was measured weekly the morning of Day 5 using von Frey filaments. In the Lieber-DeCarli model, mice received ethanol (5% v/v) or control liquid diet for 10 days, along with a single binge ethanol gavage (5 g/kg) or control gavage, respectively, on Day 10. Peripheral mechanical sensitivity was measured during the liquid diet administration and at 24 and 72 h into ethanol withdrawal. An independent group of mice that received the Lieber-DeCarli diet were administered SAHA (50 mg/kg, i.p.) during withdrawal.

Results: Male mice exhibited mechanical hypersensitivity after consuming ethanol for 5 weeks in the DID procedure. In the Lieber-DeCarli model, ethanol withdrawal led to hyperalgesia in both sexes. Suberoylanilide hydroxamic acid treatment during withdrawal from the ethanol liquid diet alleviated AWH.

Conclusions: These results demonstrate AWH in mice after chronic binge drinking in males and after Lieber-DeCarli liquid diet administration in both sexes. Like previous findings in rats, HDAC inhibition reduced AWH in mice, suggesting that epigenetic mechanisms are involved in AWH.

Keywords: alcohol withdrawal; allodynia; binge drinking; epigenetic; pain.

Carvalho L and Lasek AW. “It is not just about transcription: involvement of brain RNA splicing in substance use disorders.” ournal of neural transmission (Vienna, Austria : 1996). Publisher's Version Abstract

Alternative splicing is a co-transcriptional process that significantly contributes to the molecular landscape of the cell. It plays a multifaceted role in shaping gene transcription, protein diversity, and functional adaptability in response to environmental cues. Recent studies demonstrate that drugs of abuse have a profound impact on alternative splicing patterns within different brain regions. Drugs like alcohol and cocaine modify the expression of genes responsible for encoding splicing factors, thereby influencing alternative splicing of crucial genes involved in neurotransmission, neurogenesis, and neuroinflammation. Notable examples of these alterations include alcohol-induced changes in splicing factors such as HSPA6 and PCBP1, as well as cocaine's impact on PTBP1 and SRSF11. Beyond the immediate effects of drug exposure, recent research has shed light on the role of alternative splicing in contributing to the risk of substance use disorders (SUDs). This is exemplified by exon skipping events in key genes like ELOVL7, which can elevate the risk of alcohol use disorder. Lastly, drugs of abuse can induce splicing alterations through epigenetic modifications. For example, cocaine exposure leads to alterations in levels of trimethylated lysine 36 of histone H3, which exhibits a robust association with alternative splicing and serves as a reliable predictor for exon exclusion. In summary, alternative splicing has emerged as a critical player in the complex interplay between drugs of abuse and the brain, offering insights into the molecular underpinnings of SUDs.

Keywords: Addiction; Alcohol; Alternative splicing; Cocaine; Spliceosome; Splicing factor; Substance use disorder.

Hitzemann R, Ozburn AR, Lockwood D, and Phillips TJ. “Modeling Brain Gene Expression in Alcohol UseDisorder with Genetic Animal Models.” Current topics in behavioral neurosciences. Publisher's Version Abstract

Animal genetic models have and will continue to provide important new information about the behavioral and physiological adaptations associated with alcohol use disorder (AUD). This chapter focuses on two models, ethanol preference and drinking in the dark (DID), their usefulness in interrogating brain gene expression data and the relevance of the data obtained to interpret AUD-related GWAS and TWAS studies. Both the animal and human data point to the importance for AUD of changes in synaptic transmission (particularly glutamate and GABA transmission), of changes in the extracellular matrix (specifically including collagens, cadherins and protocadherins) and of changes in neuroimmune processes. The implementation of new technologies (e.g., cell type-specific gene expression) is expected to further enhance the value of genetic animal models in understanding AUD.

Keywords: Alcohol use disorder; Animal genetic models; Brain gene expression; Drinking in the dark; Ethanol preference; GWAS; RNA sequencing; TWAS.

Lovinger DM and Roberto M.Synaptic Effects Induced by Alcohol..” Current Topics in Behavioral Neurosciences. Publisher's Version Abstract

Ethanol (EtOH) has effects on numerous cellular molecular targets, and alterations in synaptic function are prominent among these effects. Acute exposure to EtOH activates or inhibits the function of proteins involved in synaptic transmission, while chronic exposure often produces opposing and/or compensatory/homeostatic effects on the expression, localization, and function of these proteins. Interactions between different neurotransmitters (e.g., neuropeptide effects on release of small molecule transmitters) can also influence both acute and chronic EtOH actions. Studies in intact animals indicate that the proteins affected by EtOH also play roles in the neural actions of the drug, including acute intoxication, tolerance, dependence, and the seeking and drinking of EtOH. The present chapter is an update of our previous Lovinger and Roberto (Curr Top Behav Neurosci 13:31–86, 2013) chapter and reviews the literature describing these acute and chronic synaptic effects of EtOH with a focus on adult animals and their relevance for synaptic transmission, plasticity, and behavior.

Salem NA, Manzano L, Keist MW, Ponomareva O, Roberts AJ, Roberto M, and Mayfield RD. “Cell-type brain-region specific changes in prefrontal cortex of a mouse model of alcohol dependence.” Neurobiology of disease, 190, Pp. 106361. Publisher's Version Abstract

he prefrontal cortex is a crucial regulator of alcohol drinking, and dependence, and other behavioral phenotypes associated with AUD. Comprehensive identification of cell-type specific transcriptomic changes in alcohol dependence will improve our understanding of mechanisms underlying the excessive alcohol use associated with alcohol dependence and will refine targets for therapeutic development. We performed single nucleus RNA sequencing (snRNA-seq) and Visium spatial gene expression profiling on the medial prefrontal cortex (mPFC) obtained from C57BL/6 J mice exposed to the two-bottle choice-chronic intermittent ethanol (CIE) vapor exposure (2BC-CIE, defined as dependent group) paradigm which models phenotypes of alcohol dependence including escalation of alcohol drinking. Gene co-expression network analysis and differential expression analysis identified highly dysregulated co-expression networks in multiple cell types. Dysregulated modules and their hub genes suggest novel understudied targets for studying molecular mechanisms contributing to the alcohol dependence state. A subtype of inhibitory neurons was the most alcohol-sensitive cell type and contained a downregulated gene co-expression module; the hub gene for this module is Cpa6, a gene previously identified by GWAS to be associated with excessive alcohol consumption. We identified an astrocytic Gpc5 module significantly upregulated in the alcohol-dependent group. To our knowledge, there are no studies linking Cpa6 and Gpc5 to the alcohol-dependent phenotype. We also identified neuroinflammation related gene expression changes in multiple cell types, specifically enriched in microglia, further implicating neuroinflammation in the escalation of alcohol drinking. Here, we present a comprehensive atlas of cell-type specific alcohol dependence mediated gene expression changes in the mPFC and identify novel cell type-specific targets implicated in alcohol dependence.

Keywords: Alcohol dependence; Alcohol dependence cell-type specific responses; Chronic intermittent ethanol exposure; Gene co-expression networks; Multimodal data integration; Single nucleus RNA sequencing; Spatial transcriptomics.

Torres Irizarry VC, Feng B, Yang X, Patel N, Schaul S, Ibrahimi L, Ye H, Luo P, Carrillo-Sáenz L, Lai P, Kota M, Dixit D, Wang C, Lasek AW, He Y, and Xu P. “Estrogen signaling in the dorsal raphe regulatesbinge-like drinking in mice.” Transl Psychiatry, 14, 122. Publisher's Version Abstract
Estrogens promote binge alcohol drinking and contribute to sex differences in alcohol use disorder. However, the mechanisms are largely unknown. This study aims to test if estrogens act on 5-hydroxytryptamine neurons in the dorsal raphe nucleus (5-HTDRN) to promote binge drinking. We found that female mice drank more alcohol than male mice in chronic drinking in the dark (DID) tests. This sex difference was associated with distinct alterations in mRNA expression of estrogen receptor α (ERα) and 5-HT-related genes in the DRN, suggesting a potential role of estrogen/ERs/5-HT signaling. In supporting this view, 5-HTDRN neurons from naïve male mice had lower baseline firing activity but higher sensitivity to alcohol-induced excitation compared to 5-HTDRN neurons from naïve female mice. Notably, this higher sensitivity was blunted by 17β-estradiol treatment in males, indicating an estrogen-dependent mechanism. We further showed that both ERα and ERβ are expressed in 5-HTDRNneurons, whereas ERα agonist depolarizes and ERβ agonist hyperpolarizes 5-HTDRN neurons. Notably, both treatments blocked the stimulatory effects of alcohol on 5-HTDRN neurons in males, even though they have antagonistic effects on the activity dynamics. These results suggest that ERs’ inhibitory effects on ethanol-induced burst firing of 5-HTDRN neurons may contribute to higher levels of binge drinking in females. Consistently, chemogenetic activation of ERα- or ERβ-expressing neurons in the DRN reduced binge alcohol drinking. These results support a model in which estrogens act on ERα/β to prevent alcohol-induced activation of 5-HTDRN neurons, which in return leads to higher binge alcohol drinking.
Plasil SL, Farris SP, Blednov Y, Mayfield RD, Mangieri RA, Nwokeji UJ, Aziz HC, Lambeth PS, Harris RA, and Homanics GE. “Mutation of novel ethanol-responsive lncRNA Gm41261 impacts ethanol-relatedbehavioral responses in mice.” Genes, brain, and behavior, 23, 1. Publisher's Version Abstract

Chronic alcohol exposure results in widespread dysregulation of gene expression that contributes to the pathogenesis of Alcohol Use Disorder (AUD). Long noncoding RNAs are key regulators of the transcriptome that we hypothesize coordinate alcohol-induced transcriptome dysregulation and contribute to AUD. Based on RNA-Sequencing data of human prefrontal cortex, basolateral amygdala and nucleus accumbens of AUD versus non-AUD brain, the human LINC01265 and its predicted murine homolog Gm41261 (i.e., TX2) were selected for functional interrogation. We tested the hypothesis that TX2 contributes to ethanol drinking and behavioral responses to ethanol. CRISPR/Cas9 mutagenesis was used to create a TX2 mutant mouse line in which 306 base-pairs were deleted from the locus. RNA analysis revealed that an abnormal TX2 transcript was produced at an unchanged level in mutant animals. Behaviorally, mutant mice had reduced ethanol, gaboxadol and zolpidem-induced loss of the righting response and reduced tolerance to ethanol in both sexes. In addition, a male-specific reduction in two-bottle choice every-other-day ethanol drinking was observed. Male TX2 mutants exhibited evidence of enhanced GABA release and altered GABAA receptor subunit composition in neurons of the nucleus accumbens shell. In C57BL6/J mice, TX2 within the cortex was cytoplasmic and largely present in Rbfox3+ neurons and IBA1+ microglia, but not in Olig2+ oligodendrocytes or in the majority of GFAP+ astrocytes. These data support the hypothesis that TX2 mutagenesis and dysregulation impacts ethanol drinking behavior and ethanol-induced behavioral responses in mice, likely through alterations in the GABAergic system.

Keywords: CRISPR/Cas9; alcohol use disorder; behavioral analysis; electrophysiology; gene-targeted; genetics; long noncoding RNA; molecular analysis; mouse; mutagenesis.

Mason BJ, Estey D, Roberts A, Guglielmo de G, George O, Light J, Stoolmiller M, Quello S, Skinner M, Shadan F, Begovic A, Kyle MC, and Harris RA. “A reverse translational study of PPAR-α agonist efficacy inhuman and rodent models relevant to alcohol use disorder..” Neurobiology of stress, 29, 100604. Publisher's Version Abstract

Alcohol Use Disorder (AUD) is a chronic relapsing disorder affecting an estimated 283 million individuals worldwide, with substantial health and economic consequences. Peroxisome proliferator-activated receptors (PPARs), particularly PPAR-α and PPAR-γ, have shown promise in preclinical studies as potential therapeutic targets for AUD. In this human laboratory study, we aimed to translate preclinical findings on the PPAR-α agonist fenofibrate to a human population with current AUD. We hypothesized that, relative to placebo, fenofibrate at the highest FDA-approved dose of 145 mg/d would attenuate responsiveness to in vivo alcohol cues in the lab and reduce drinking under natural conditions. However, the results did not show significant differences in craving and alcohol consumption between the fenofibrate and placebo groups. Reverse translational studies in rodent models confirmed the lack of fenofibrate effect at human-equivalent doses. These findings suggest that inadequate translation of drug dose from rodents to humans may account for the lack of fenofibrate effects on alcohol craving and consumption in humans with AUD. The results highlight the need for new brain-penetrant PPAR-α agonists to adequately test the therapeutic potential of PPAR-α agonists for AUD, and the importance of reverse translational approaches and selection of human-equivalent doses in drug development.

Keywords: Alcohol use disorder; Fenofibrate; Human laboratory study; Mouse; Peroxisome proliferator-activated receptor-alpha; Rat.

Ozburn AR and Spencer SM.Repurposing anti-inflammatory medications for alcohol and substance use disorders.” Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. Publisher's Version
Grigsby KB, Mangieri RA, Roberts AJ, Lopez MF, Firsick EJ, Townsley KG, Beneze A, Bess J, Eisenstein TK, Meissler JJ, Light JM, Miller J, Quello S, Shadan F, Skinner MH, Aziz HC, Metten P, Morissett RA, Crabbe JC, Roberto M, Becker HC, Mason BJ, and Ozburn AR.Pre-clinical and clinical evidence forsuppression of alcohol intake by apremilast..” The Journal of clinical investigation, 133, 6, Pp. e159103. Publisher's Version Abstract
Treatment options for Alcohol Use Disorders (AUD) have minimally advanced since 2004, while the annual deaths and economic toll have increased alarmingly. Phosphodiesterase type 4 (PDE4) is associated with alcohol and nicotine dependence. PDE4 inhibitors were identified as a potential AUD treatment using a novel bioinformatics approach. We prioritized a newer PDE4 inhibitor, apremilast, as ideal for repurposing, (i.e. FDA approved for psoriasis, low incidence of adverse events, excellent safety profile), and tested it using multiple animal strains and models, as well as in a human Phase IIa study. We found that apremilast reduced binge-like alcohol intake and behavioral measures of alcohol motivation in mouse models of genetic risk for drinking to intoxication. Apremilast also reduced excessive alcohol drinking in models for stress-facilitated drinking and alcohol dependence. Using site-directed drug infusions and electrophysiology, we uncovered that apremilast may act to lessen drinking in mice by increasing neural activity in the nucleus accumbens, a key brain region in the regulation of alcohol intake. Importantly, apremilast (90 mg/d) reduced excessive drinking in non-treatment seeking individuals with AUD in a double blind, placebo-controlled study. These results demonstrate that apremilast suppresses excessive alcohol drinking across the spectrum of AUD severity.
Athanason AC, Nadav T, Cates-Gatto C, Roberts AJ, Roberto M, and Varodayan FP.Chronic ethanol altersadrenergic receptor gene expression and produces cognitive deficits in male mice..” Neurobiology of stress, 24, Pp. 100542. Publisher's Version Abstract
Hyperkateifia and stress-induced alcohol cravings drive relapse in individuals with alcohol use disorder (AUD). The brain stress signal norepinephrine (also known as noradrenaline) tightly controls cognitive and affective behavior and was thought to be broadly dysregulated with AUD. The locus coeruleus (LC) is a major source of forebrain norepinephrine, and it was recently discovered that the LC sends distinct projections to addiction-associated regions suggesting that alcohol-induced noradrenergic changes may be more brain region-specific than originally thought. Here we investigated whether ethanol dependence alters adrenergic receptor gene expression in the medial prefrontal cortex (mPFC) and central amgydala (CeA), as these regions mediate the cognitive impairment and negative affective state of ethanol withdrawal. We exposed male C57BL/6J mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and assessed reference memory, anxiety-like behavior and adrenergic receptor transcript levels during 3-6 days of withdrawal. Dependence bidirectionally altered mouse brain α1 and β receptor mRNA levels, potentially leading to reduced mPFC adrenergic signaling and enhanced noradrenergic influence over the CeA. These brain region-specific gene expression changes were accompanied by long-term retention deficits and a shift in search strategy in a modified Barnes maze task, as well as greater spontaneous digging behavior and hyponeophagia. Current clinical studies are evaluating adrenergic compounds as a treatment for AUD-associated hyperkatefia, and our findings can contribute to the refinement of these therapies by increasing understanding of the specific neural systems and symptoms that may be targeted.
Varodayan FP, Pahng AR, Davis TD, Gandhi P, Bajo M, Steinman MQ, Kiosses WB, Blednov YA, Burkart MD, Edwards S, Roberts AJ, and Roberto M.Chronic ethanol induces a pro-inflammatory switch ininterleukin-1β regulation of GABAergic signaling in the medial prefrontal cortex of male mice..” Brain, behavior, and immunity, 110, Pp. 125–139. Publisher's Version Abstract
Neuroimmune pathways regulate brain function to influence complex behavior and play a role in several neuropsychiatric diseases, including alcohol use disorder (AUD). In particular, the interleukin-1 (IL-1) system has emerged as a key regulator of the brain's response to ethanol (alcohol). Here we investigated the mechanisms underlying ethanol-induced neuroadaptation of IL-1β signaling at GABAergic synapses in the prelimbic region of the medial prefrontal cortex (mPFC), an area responsible for integrating contextual information to mediate conflicting motivational drives. We exposed C57BL/6J male mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and conducted ex vivo electrophysiology and molecular analyses. We found that the IL-1 system regulates basal mPFC function through its actions at inhibitory synapses on prelimbic layer 2/3 pyramidal neurons. IL-1β can selectively recruit either neuroprotective (PI3K/Akt) or pro-inflammatory (MyD88/p38 MAPK) mechanisms to produce opposing synaptic effects. In ethanol naïve conditions, there was a strong PI3K/Akt bias leading to a disinhibition of pyramidal neurons. Ethanol dependence produced opposite IL-1 effects - enhanced local inhibition via a switch in IL-1β signaling to the canonical pro-inflammatory MyD88 pathway. Ethanol dependence also increased cellular IL-1β in the mPFC, while decreasing expression of downstream effectors (Akt, p38 MAPK). Thus, IL-1β may represent a key neural substrate in ethanol-induced cortical dysfunction. As the IL-1 receptor antagonist (kineret) is already FDA-approved for other diseases, this work underscores the high therapeutic potential of IL-1 signaling/neuroimmune-based treatments for AUD.
Lasek AW, da Silva D, and Choi DS.Editorial: Molecular aspects of compulsive drug use.” Frontiers in psychiatry, 14. Publisher's Version Abstract
Drug addiction is a complex psychiatric disorder defined by a compulsion to seek and take the drug, losing control over intake, and continuing to take the drug despite negative consequences. The inability to limit drug consumption leads to relapse and failure in treatment, thus understanding the underlying molecular mechanisms that contribute to compulsive drug seeking and taking is critical to developing effective treatments. The medical term for drug addiction is substance use disorder (SUD), which is defined by an individual having two or more of the 11 criteria that are outlined in the 5th edition of the American Psychiatric Association (1) Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Several criteria for SUD encompass a wide range of indicators that associate with compulsive drug use. These criteria specifically focus on two key aspects: risky use and social impairment. Risky use is characterized by recurrent substance use in physically unsafe environments and persistent substance use despite awareness of potential physical or psychological harm. Social impairment criteria encompass the inability to fulfill major obligations at work, school, or home; continued use of the substance despite significant social or interpersonal problems; and reduction or discontinuation of recreational, social, or occupational activities due to substance use. Despite significant research efforts, the precise molecular mechanisms underlying compulsive drug use in remain largely unknown. However, gaining a deeper understanding of the neurobiological mechanisms that underly compulsive drug taking could lead to novel pharmacological and psychological tools to treat SUD. The papers in this Research Topic, “Molecular aspects of compulsive drug use” provide new knowledge toward achieving this goal. This collection contains preclinical and clinical studies on three different substances: alcohol, cocaine, and methamphetamine. Three of the papers on this topic also have a secondary emphasis on sex differences in compulsive drug use. Women have historically been underrepresented in behavioral neuroscience research but like men, also suffer from SUD. Understanding differences in neurobiology that contribute to sex differences in compulsive drug use will aid in developing effective treatments for both sexes.
The role astrocytes play in brain development and function has garnered greater attention as the diversity of roles they are involved in has become apparent. We have previously shown that ethanol-exposed astrocytes alter neuronal neurite outgrowth in an in vitro co-culture system and that ethanol alters the astrocyte-produced extracellular matrix (ECM) in vitro, with similar alterations in vivo. In this study, we utilized the translating ribosome affinity purification (TRAP) procedure in Aldh1l1-EGFP/Rpl10a transgenic mouse primary cortical astrocyte cultures to transcriptionally and translationally profile the astrocyte response to ethanol. We found a large number of differences between the total RNA pool and the translating RNA pool, indicating that the transcriptional state of astrocytes may not always reflect the translational state of astrocytes. In addition, there was a considerable overlap between ethanol-dysregulated genes in the total RNA pool and the translating RNA pool. Comparisons to published datasets indicate the in vitro model used here is most similar to PD1 or PD7 in vivo cortical astrocytes, and the ethanol-regulated genes showed a significant overlap with models of chronic ethanol exposure in astrocytes, a model of third-trimester ethanol exposure in the hippocampus and cerebellum, and an acute model of ethanol exposure in the hippocampus. These findings will further our understanding of the effects of ethanol on astrocyte gene expression and protein translation and how these changes may alter brain development and support the use of in vitro astrocyte cultures as models of neonatal astrocytes.
Borgonetti V, Cruz B, Vozella V, Khom S, Steinman MQ, Bullard R, D'Ambrosio S, Oleata CS, Vlkolinsky R, Bajo M, Zorrilla EP, Kirson D, and Roberto M.IL-18 Signaling in the Rat Central Amygdala Is Disrupted in a Comorbid Model of Post-Traumatic Stress and Alcohol Use Disorder.” Cells, 12, 15. Publisher's Version Abstract

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share dysregulated neuroimmune-related pathways. Here, we used our established rat model of comorbid post-traumatic stress disorder (PTSD)/AUD to characterize the interleukin 18 (IL-18) system in the central amygdala (CeA). Male and female rats underwent novel (NOV) and familiar (FAM) shock stress, or no stress (unstressed controls; CTL) followed by voluntary alcohol drinking and PTSD-related behaviors, then all received renewed alcohol access prior to the experiments. In situ hybridization revealed that the number of CeA positive cells for Il18 mRNA increased, while for Il18bp decreased in both male and female FAM stressed rats versus CTL. No changes were observed in Il18r1 expression across groups. Ex vivo electrophysiology showed that IL-18 reduced GABAA-mediated miniature inhibitory postsynaptic currents (mIPSCs) frequencies in CTL, suggesting reduced CeA GABA release, regardless of sex. Notably, this presynaptic effect of IL-18 was lost in both NOV and FAM males, while it persisted in NOV and FAM females. IL-18 decreased mIPSC amplitude in CTL female rats, suggesting postsynaptic effects. Overall, our results suggest that stress in rats with alcohol access impacts CeA IL-18-system expression and, in sex-related fashion, IL-18′s modulatory function at GABA synapses.


IL-18post-traumatic stress disorderalcohol use disorderCeAsex differencesGABA

Alcohol consumption activates the neuroimmune system of the brain, a system in which brain astrocytes and microglia play dominant roles. These glial cells normally produce low levels of neuroimmune factors, which are important signaling factors and regulators of brain function. Alcohol activation of the neuroimmune system is known to dysregulate the production of neuroimmune factors, such as the cytokine IL-6, thereby changing the neuroimmune status of the brain, which could impact the actions of alcohol. The consequences of neuroimmune–alcohol interactions are not fully known. In the current studies we investigated this issue in transgenic (TG) mice with altered neuroimmune status relative to IL-6. The TG mice express elevated levels of astrocyte-produced IL-6, a condition known to occur with alcohol exposure. Standard behavioral tests of alcohol drinking and negative affect/emotionality were carried out in homozygous and heterozygous TG mice and control mice to assess the impact of neuroimmune status on the actions of chronic intermittent alcohol (ethanol) (CIE) exposure on these behaviors. The expressions of signal transduction and synaptic proteins were also assessed by Western blot to identify the impact of alcohol–neuroimmune interactions on brain neurochemistry. The results from these studies show that neuroimmune status with respect to IL-6 significantly impacts the effects of alcohol on multiple levels.


neuroimmunesynaptic transmissionalcohol drinkingdepressive-like behaviorSTAT3p42/44MAPKGABAAR subunits

Rice RC, Gil DV, Baratta AM, Frawley RR, Hill SY, Farris SP, and Homanics GE. “Inter- and transgenerationalheritability of preconception chronic stress or alcohol exposure: Translational outcomes in brain andbehavior..” Neurobiology of stress, 29, 100603. Publisher's Version Abstract

Chronic stress and alcohol (ethanol) use are highly interrelated and can change an individual's behavior through molecular adaptations that do not change the DNA sequence, but instead change gene expression. A recent wealth of research has found that these nongenomic changes can be transmitted across generations, which could partially account for the "missing heritability" observed in genome-wide association studies of alcohol use disorder and other stress-related neuropsychiatric disorders. In this review, we summarize the molecular and behavioral outcomes of nongenomic inheritance of chronic stress and ethanol exposure and the germline mechanisms that could give rise to this heritability. In doing so, we outline the need for further research to: (1) Investigate individual germline mechanisms of paternal, maternal, and biparental nongenomic chronic stress- and ethanol-related inheritance; (2) Synthesize and dissect cross-generational chronic stress and ethanol exposure; (3) Determine cross-generational molecular outcomes of preconception ethanol exposure that contribute to alcohol-related disease risk, using cancer as an example. A detailed understanding of the cross-generational nongenomic effects of stress and/or ethanol will yield novel insight into the impact of ancestral perturbations on disease risk across generations and uncover actionable targets to improve human health.

Keywords: Alcohol; Epigenetics; Inheritance; Intergenerational; Stress; Transgenerational.

Zhu L, Brown MA, Sims RJ, Tiwari GR, Nie H, Mayfield RD, and Tucker HO.Lysine Methyltransferase SMYD1 Regulates Myogenesis via skNAC Methylation..” Cells, 12, 13. Publisher's Version Abstract

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin (Mb), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.

Keywords: heart and skeletal muscle; methyltransferase; transcriptional regulation.

Rodd ZA, Swartzwelder HS, Waeiss RA, Soloviov SO, Lahiri DK, Engleman EA, Truitt WA, Bell RL, and Hauser SR.Negative and positive allosteric modulators of the α7 nicotinic acetylcholine receptor regulates theability of adolescent binge alcohol exposure to enhance adult alcohol consumption..” Frontiers in behavioral neuroscience, 16, Pp. 954319. Publisher's Version Abstract
Rationale and Objectives: Ethanol acts directly on the α7 Nicotinic acetylcholine receptor (α7). Adolescent-binge alcohol exposure (ABAE) produces deleterious consequences during adulthood, and data indicate that the α7 receptor regulates these damaging events. Administration of an α7 Negative Allosteric Modulator (NAM) or the cholinesterase inhibitor galantamine can prophylactically prevent adult consequences of ABAE. The goals of the experiments were to determine the effects of co-administration of ethanol and a α7 agonist in the mesolimbic dopamine system and to determine if administration of an α7 NAM or positive allosteric modulator (PAM) modulates the enhancement of adult alcohol drinking produced by ABAE. Methods: In adult rats, ethanol and the α7 agonist AR-R17779 (AR) were microinjected into the posterior ventral tegmental area (VTA), and dopamine levels were measured in the nucleus accumbens shell (AcbSh). In adolescence, rats were treated with the α7 NAM SB-277011-A (SB) or PNU-120596 (PAM) 2 h before administration of EtOH (ABAE). Ethanol consumption (acquisition, maintenance, and relapse) during adulthood was characterized. Results: Ethanol and AR co-administered into the posterior VTA stimulated dopamine release in the AcbSh in a synergistic manner. The increase in alcohol consumption during the acquisition and relapse drinking during adulthood following ABAE was prevented by administration of SB, or enhanced by administration of PNU, prior to EtOH exposure during adolescence. Discussion: Ethanol acts on the α7 receptor, and the α7 receptor regulates the critical effects of ethanol in the brain. The data replicate the findings that cholinergic agents (α7 NAMs) can act prophylactically to reduce the alterations in adult alcohol consumption following ABAE.