Alcohol use disorder (AUD) is one of the most prevalent mental health disorders worldwide yet effective therapeutics remain limited. Mounting evidence indicates that dysregulated immune signaling in the brain plays a role in AUD pathophysiology. Activation of pro-inflammatory pathways like the interleukin-6 (IL-6) pathway represents a potential point of convergence between synaptic dysfunction and motivational changes in AUD that remain undiscovered. Thus, using a translational neuroscience approach and well-established model of chronic alcohol intake, we investigated the cell-type specific role of IL-6 signaling in the central amygdala, a critical region in the development and maintenance of alcohol dependence. We demonstrate that chronic alcohol exposure recruits IL-6-related pathways in humans and rodents via astrocytic, neuronal, and microglial mechanisms, and that IL-6 inhibits central amygdala GABAergic transmission. Notably, systemic administration of an IL-6 receptor antibody decreased alcohol drinking in alcohol-dependent female mice. Collectively, our findings support IL-6 inhibition as a novel-neuroimmune-targeted therapeutic strategy to reduce excessive drinking in the context of AUD.

Astrocytes play essential roles in maintaining brain homeostasis and in contributing to synaptic functions, but, in response to injury, infection, or disease, astrocytes can downregulate their homeostatic and physiological functions while increasing neuroinflammatory responses. The central amygdala (CeA) is important for stress responsivity and the development of alcohol (ethanol) dependence. Using a multi-omics approach in Aldh1l1-EGFP/Rpl10a mice and the chronic intermittent ethanol two-bottle choice (CIE-2BC) model, we have characterized the translational response of CeA astrocytes, as well as the proteomic and phosphoproteomic changes in ethanol dependent, non-dependent, and naïve mice. We identified astrocyte-specific alterations in neuroimmune functions and antioxidant/oxidative stress pathways in ethanol dependent mice as well as cytoskeletal plasticity related pathways in non-dependent mice. Proteomic analysis showed down-regulation of astrocyte physiological functions in dependent animals while phosphoproteomic analysis identified pathways associated with cytoskeleton remodeling in both dependent and non-dependent mice. Reconstructions of astrocyte morphologies demonstrated increased CeA astrocyte complexity in dependent and non-dependent groups compared to naïve mice. The astrocyte-specific activation of neuroimmune and antioxidant pathways, down-regulation of homeostatic functions, alteration in protein phosphorylation-mediated cytoskeleton remodeling, and increased astrocyte morphological complexity demonstrate that ethanol dependence induces astrocyte reactivity in the CeA consistent with both adaptive and maladaptive changes. These findings highlight the role of CeA astrocytes in the progression from alcohol intake to dependence and represent a first step toward identifying astrocyte-specific therapeutic strategies to treat Alcohol Use Disorder (AUD) aimed at potentiating reactive astrocyte adaptive changes and inhibiting maladaptive responses.

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease, yet mass spectrometry-based histone PTM analysis remains constrained by inefficient sample preparation workflows. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multiprotease workflow that reduces sample preparation to hours while improving PTM coverage and quantitative accuracy. Systematic evaluation of Arg-C Ultra and a recombinant (r)-Chymotrypsin protease under varied conditions, including standard derivatization with propionic anhydride and tandem mass tag (TMT) labeling, demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM that exceeds Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMT's tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 58 succinylation and 31 glutarylation sites─a "dark epigenome" largely undetected by propionylation-based methods. Complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling the detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. In HEK293T cells treated with the pan-sirtuin inhibitor nicotinamide, RIPUP quantified 112 statistically significant peptidoforms (adj p < 0.05), predominantly increasing with the NAM dose (88 up, 24 down). Application of RIPUP to frozen-thawed rat hippocampal sections within a 3 h workflow identified >200 PTMs including H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A K118/K119 ubiquitination. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.

Alcohol use disorder (AUD) is a complex polygenic disease. Rodent models of alcohol dependence have been instrumental in modeling various aspects of dependence. Single-nucleus transcriptomics has enabled the profiling of cell-type-specific changes in gene expression in both human AUD and animal models. In this study, we identified shared dysregulated transcriptomic networks (TN), comprising gene co-expression modules and gene regulatory networks (GRNs) in a mouse model of alcohol dependence and individuals with AUD. Through cell-type-specific TN analysis, we identified translationally relevant, conserved dependence dysregulated molecular signatures. We identified conserved dependence-upregulated gene co-expression modules in astrocytes and oligodendrocytes, with hub genes Slc1a3 and Pde4b, respectively. These genes are linked to alcohol dependence mechanisms, such as glutamate signaling, a well-established target of alcohol’s effects, and PDE4, whose inhibition has been shown to reduce alcohol intake in preclinical and clinical studies. We then integrated publicly available human and mouse GRN data to identify upstream regulators of alcohol-dysregulated gene signatures in each cell type. This approach revealed a set of transcription factors (TFs), including Mef2a, Mef2c, Jund, Nr3c1, and Zeb1, that were upstream of most dysregulated genes in both the mouse and human datasets and have established relevance to addiction biology, representing promising targets for translational research. Collectively, these findings demonstrate the utility of cross-species, cell-type-specific network analysis for uncovering conserved molecular mechanisms in alcohol dependence. The identification of shared dysregulated networks, cell type homology, and upstream regulators provides a foundation for developing translationally relevant targeting strategies that can be tested in animal models.

Abstract Astrocytes play an important role in neuronal health. A critical function of astrocytes is to clear excess extracellular glutamate and prevent excitotoxicity. STAT3 is a transcription factor that promotes astrocyte development and astrocyte reactivity in neurodegenerative diseases and following central nervous system injury. To determine the innate molecular and behavioral functions of adult astrocyte-expressed STAT3 in a non-pathological state, we created conditional Stat3 astrocyte knockout mice (Stat3 aKO) using Stat3 flox and the tamoxifen-activated Cre line, Aldh1l1-Cre/ERT2. We measured transcript levels of Gfap , a known STAT3 target gene, and glutamate transporter genes in the medial prefrontal cortex (PFC) of Stat3 aKO. Gfap , Slc1a2 and Slc17a8 transcripts were decreased in the PFC of Stat3 aKO of both sexes. GLT-1 protein, encoded by Slc1a2 , was also reduced in the PFC of male Stat3 aKO. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) in male Stat3 aKO and control prelimbic pyramidal neurons and found increased sEPSC amplitude, consistent with a hyper-glutamatergic state due to impaired glutamate clearance. To determine the behavioral consequences of STAT3 depletion in astrocytes, Stat3 aKO were tested for locomotor activity, anxiety-like behavior and binge ethanol consumption, behaviors linked to dysregulation of glutamate homeostasis. Stat3 aKO mice did not differ in locomotor activity or anxiety-like behavior; however, male Stat3 aKO mice consumed significantly less ethanol than controls. These results indicate that STAT3 in adult astrocytes is crucial for maintaining glutamate transporter levels in the adult brain and that astrocytic STAT3 promotes ethanol consumption in male mice. Main points Gfap , Slc1a2 and Slc17a8 expression are lower in the cortex of Stat3 astrocyte knockout mice (Stat3 aKO) GLT-1 protein is decreased and glutamate neurotransmission is elevated in the cortex of male Stat3 aKO Male Stat3 aKO consume less ethanol

Single-cell proteomics (SCP) is a powerful method for interrogating the molecular composition of neurons, yet its application to acute brain slices has remained limited. Patch-clamp electrophysiology provides direct information on neuronal excitability, synaptic inputs, and ion channel function, making it a natural partner for SCP. However, combining these techniques introduces unique challenges. For instance, after patch-clamping a neuron, its soma must be physically retrieved, and variability during extraction from the brain slice may influence how well proteomic measurements reflect in situ physiology. Here, we introduce a framework for contextualizing patch-SCP outcomes, with an emphasis on retrieval quality (material yield and soma-enriched synaptic content). We used an indiscriminate shotgun strategy in which all patched neurons were collected regardless of electrophysiological outcome to assess soma retrieval in an exploratory data set of rat medial prefrontal cortex pyramidal neurons. Capacitance during gigaseal-preserved retrieval correlated with protein identifications, suggesting that proteome yield could be linked to soma size. Preservation of neuronal spiking during relocation tended to be associated with broader synaptic enrichment and recovery of transmembrane proteins. By comparison, torn or aspirated neurons produced small proteomes with poor synaptic representation and neurons with little to no characterization displayed more variable outcomes. These results demonstrate that patch-SCP can be used to assess soma retrieval and they provide a framework for interpreting how electrophysiological context and soma retrieval quality shape single-neuron proteomic measurements in semi-intact circuits.

The neurocircuitry changes mediating the development and maintenance of an alcohol use disorder are complex and dynamic. The parasubthalamic nucleus (PSTN), a small nucleus of the posterior lateral hypothalamus best known for suppressing appetite, is interconnected with brain regions disrupted in addiction; yet its potential role in the regulation of alcohol consumption had never been examined. Here we show that the PSTN exerts potent control over alcohol drinking in mice. Remarkably, the influence of endogenous PSTN activity on voluntary alcohol consumption switches from inhibitory to stimulatory upon induction of alcohol dependence. Among PSTN cells, Crh neurons represent a unique subpopulation that promotes alcohol drinking and fires more in dependent mice. Alcohol intake escalation driven by PSTN Crh neurons involves thalamic output and behavioral disinhibition. Based on our results, PSTN Crh neurons could represent a critical node in the brain circuitry overactive in alcohol addiction driven by reward seeking in humans.

Alcohol use disorder (AUD) is defined by the emergence of negative affective symptoms during withdrawal. Neuroinflammation is a key contributor to AUD, and it is well known to play an essential role in the pathogenesis of pain states. The chronic-intermittent ethanol two-bottle choice (CIE-2BC) paradigm is well-established to generate alcohol-dependent (Dep) and non-dependent (Non-Dep) mice. Our recent work demonstrated that the CIE-2BC model promotes mechanical allodynia in Dep mice, with these mice developing mechanical allodynia during withdrawal. In this study, we examined the role of interleukin-6 (IL-6) in the development of mechanical allodynia by adapting the CIE-2BC mouse model and employing the von Frey test, in situ hybridization (RNAscope), and Multiplex protein analysis of the spinal cord, examining changes in this target including an array of cytokines associated with IL-6 signaling. CIE-2BC escalated alcohol drinking and enhanced mechanical allodynia in Dep versus Non-Dep mice, with Dep females displaying greater alcohol intake. Dep mice displayed increased IL-6 protein in the spinal cord while males additionally had increased Il6+ cell expression versus Non-Dep controls. Systemic treatment of an IL-6 receptor antibody (IL-6R Ab) did not decrease mechanical allodynia during abstinence. Collectively, these data suggest that alcohol exerts sex-dependent effects on spinal IL-6 levels. However, in our study, blocking IL-6 signaling did not reduce alcohol-associated pain sensitivity in a mouse model of comorbid pain and alcohol dependence.

Anxiety is a major symptom associated with alcohol withdrawal and a major factor increasing the risk of relapse. Although fluoxetine, a selective serotonin reuptake inhibitor, is used to alleviate these symptoms, its effects on brain lipid signaling pathways involved in withdrawal-related anxiety remain unclear. This study evaluated, in a preclinical model, the behavioral and molecular effects of chronic alcohol exposure and fluoxetine treatment during early abstinence. Male Wistar rats received oral alcohol (3 g/kg) or saline for 14 days, followed by 7 days without alcohol, during which fluoxetine (10 mg/kg) was administered to designated groups. Anxiety-like behavior was assessed using the elevated plus maze. Circulating plasma levels of corticosterone, 2-arachidonoylglycerol (2-AG), lysophosphatidic acid (LPA), and interleukin-10 (IL-10) were quantified, and gene expression analyses were performed in the amygdala and medial prefrontal cortex (mPFC). Chronic alcohol administration increased anxiety-like behavior and plasma 2-AG, while reducing LPA and IL-10 levels. Fluoxetine induced an anxiolytic effect in controls but was ineffective in alcohol-exposed rats, only normalizing the alcohol-induced increase of plasma 2-AG. At the molecular level, fluoxetine modulated gene expression region-specifically, altering 2-AG-related genes in the amygdala and enhancing LPA signaling in the mPFC. Hierarchical clustering revealed coordinated downregulation of 2-AG pathway genes in the alcohol-fluoxetine group and partial restoration of anti-inflammatory markers. These findings indicate fluoxetine modulates lipid signaling and immune-related genes during alcohol withdrawal, but its anxiolytic efficacy may be limited after alcohol exposure. These findings may contribute to the development of targeted therapeutic strategies for alcohol-related anxiety and relapse prevention.