Protein kinase C epsilon (PKCε) regulates behavioural responses to ethanol and plays a role in anxiety-like behaviour, but knowledge is limited on downstream substrates of PKCε that contribute to these behaviours. We recently identified brain-specific serine/threonine-protein kinase 1 (BRSK1) as a substrate of PKCε. Here, we test the hypothesis that BRSK1 mediates responses to ethanol and anxiety-like behaviours that are also PKCε dependent. We used in vitro kinase assays to further validate BRSK1 as a substrate of PKCε and used Brsk1-/- mice to assess the role of BRSK1 in ethanol- and anxiety-related behaviours and in physiological responses to ethanol. We found that BRSK1 is phosphorylated by PKCε at a residue identified in a chemical genetic screen of PKCε substrates in mouse brain. Like Prkce-/- mice, male and female Brsk1-/- mice were more sensitive than wild-type to the acute sedative-hypnotic effect of alcohol. Unlike Prkce-/- mice, Brsk1-/- mice responded like wild-type to ataxic doses of ethanol. Although in Prkce-/- mice ethanol consumption and reward are reduced in both sexes, they were reduced only in female Brsk1-/- mice. Ex vivo slice electrophysiology revealed that ethanol-induced facilitation of GABA release in the central amygdala was absent in male Brsk1-/- mice similar to findings in male Prkce-/- mice. Collectively, these results indicate that BRSK1 is a target of PKCε that mediates some PKCε-dependent responses to ethanol in a sex-specific manner and plays a role distinct from PKCε in anxiety-like behaviour.

Keywords: alcohol; anxiety; protein kinase c epsilon.

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.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed “open-access”) was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10−7) and plasma membrane changes (FDR < 1 × 10−7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10−5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

KEYWORDS: rhesus macaque, abstinence from alcohol, prefrontal cortex, RNA-seq, ethanol

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.

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.

Keywords: Acute functional tolerance to ethanol; Diazepam; Poly(I:C); Rotarod ataxia; Tlr3 knockdown in dorsal striatum; Tlr3(F/F) mice; Two-bottle choice every-other-day ethanol consumption.

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.

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.

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Keywords: Alcohol use disorder; IRF7; Neuroimmune signaling; R837; R848; Small noncoding RNAs; Toll-like receptor 7.

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.

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.

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.