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AbstractBackgroundBinge alcohol exposure during adolescence results in long‐lasting alterations in the brain and behavior. For example, adolescent intermittent ethanol (AIE) exposure in rodents results in long‐term loss of functional connectivity among prefrontal cortex (PFC) and striatal regions as well as a variety of neurochemical, molecular, and epigenetic alterations. Interneurons in the PFC and striatum play critical roles in behavioral flexibility and functional connectivity. For example, parvalbumin (PV) interneurons are known to contribute to neural synchrony and cholinergic interneurons contribute to strategy selection. Furthermore, extracellular perineuronal nets (PNNs) that surround some interneurons, particularly PV+ interneurons, further regulate cellular plasticity. The effect of AIE exposure on the expression of these markers within the PFC is not well understood.MethodsThe present study tested the hypothesis that AIE exposure reduces the expression of PV+ and choline acetyltransferase (ChAT)+ interneurons in the adult PFC and striatum and increases the related expression of PNNs (marked by binding of Wisteria floribunda agglutinin lectin) in adulthood. Male rats were exposed to AIE (5 g/kg/day, 2‐days‐on/2‐days‐off, i.e., P25 to P54) or water (CON), and brain tissue was harvested in adulthood (>P80). Immunohistochemistry and co‐immunofluorescence were used to assess the expression of ChAT, PV, and PNNs within the adult PFC and striatum following AIE exposure.ResultsChAT and PV interneuron densities in the striatum and PFC were unchanged after AIE exposure. However, PNN density in the PFC of AIE‐exposed rats was greater than in CON rats. Moreover, significantly more PV neurons were surrounded by PNNs in AIE‐exposed subjects than controls in both PFC subregions assessed: orbitofrontal cortex (CON = 34%; AIE = 40%) and medial PFC (CON = 10%; AIE = 14%).ConclusionsThese findings indicate that, following AIE exposure, PV interneuron expression in the adult PFC and striatum is unaltered, while PNNs surrounding these neurons are increased. This increase in PNNs may restrict the plasticity of the ensheathed neurons, thereby contributing to impaired microcircuitry in frontostriatal connectivity and related behavioral impairments.

Aldosterone regulates electrolyte and fluid homeostasis through binding to the mineralocorticoid receptors (MRs). Previous work provides evidence for a role of aldosterone in alcohol use disorders (AUDs). We tested the hypothesis that high functional activity of the mineralocorticoid endocrine pathway contributes to vulnerability for AUDs. In Study 1, we investigated the relationship between plasma aldosterone levels, ethanol self-administration and the expression of CYP11B2 and MR (NR3C2) genes in the prefrontal cortex area (PFC) and central nucleus of the amygdala (CeA) in monkeys. Aldosterone significantly increased after 6- and 12-month ethanol self-administration. NR3C2 expression in the CeA was negatively correlated to average ethanol intake during the 12 months. In Study 2, we measured Nr3c2 mRNA levels in the PFC and CeA of dependent and nondependent rats and the correlates with ethanol drinking during acute withdrawal. Low Nr3c2 expression levels in the CeA were significantly associated with increased anxiety-like behavior and compulsive-like drinking in dependent rats. In Study 3, the relationship between plasma aldosterone levels, alcohol drinking and craving was investigated in alcohol-dependent patients. Non-abstinent patients had significantly higher aldosterone levels than abstinent patients. Aldosterone levels positively correlated with the number of drinks consumed, craving and anxiety scores. These findings support a relationship between ethanol drinking and the aldosterone/MR pathway in three different species.

AbstractThe limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.

GABA(A) receptors are sensitive to ethanol in distinct brain regions and are clearly involved in the acute actions of ethanol, ethanol tolerance, ethanol dependence and ethanol self-administration. Data from a variety of perspectives such as molecular, cellular and behavioral analysis have elucidated the role of GABA(A) receptors in these processes. GABA(A) receptor activation mediates many of the behavioral effects of ethanol including motor incoordination, anxiolysis and sedation. The actions of ethanol at GABA(A) receptors are influenced by endogenous modulators such as the neuroactive steroids. Sensitization to these compounds influences ethanol dependence and withdrawal and may explain gender differences in the molecular effects of ethanol. Furthermore, GABA(A) receptors may also play a role in ethanol self-administration via the mesolimbic reward system. Ethanol tolerance and dependence may be explained, in part, by changes in the function of GABA(A) receptors. We have proposed that alterations in native GABA(A) receptor subunit assembly could alter the functional properties of these receptors. However, post-translational modifications or other post-synaptic mechanisms may also explain changes in GABA(A) receptor function. Genetic animal models of ethanol dependence have also identified GABA(A) receptor genes as likely mediators of the behavioral adaptations associated with ethanol dependence and withdrawal. A better understanding of the effects of ethanol at GABA(A) receptors has highlighted important potential mechanisms involved in the development of alcoholism.

GABA(A) receptors are involved in the subjective effects of alcohol. Endogenous neuroactive steroids interact with GABA(A) receptors to mediate several behavioral effects of alcohol in rodents. Based on a haplotypic association of alcohol dependence with the gene encoding the GABA(A) receptor alpha-2 subunit (GABRA2), we examined whether GABRA2 alleles are associated with the subjective response to alcohol. We also examined whether finasteride (a 5-alpha steroid reductase inhibitor), which blocks the synthesis of some neuroactive steroids, reduces the subjective response to alcohol. In all, 27 healthy social drinkers (15 males) completed a randomized, double-blind, placebo-controlled study of high-dose finasteride. After being pretreated with study drug, subjects consumed three alcoholic drinks. Subjective effects were measured repeatedly over the ascending blood alcohol curve. To examine the moderating role of genetic variation in GABRA2, a single-nucleotide polymorphism that was informative in association studies was included as a factor in the analysis. Subjects homozygous for the more common A-allele (n=7) showed more subjective effects of alcohol than did individuals with one or two copies of the alcohol dependence-associated G-allele (n=20, including two homozygotes). Among the A-allele homozygotes, there was a greater reduction in several subjective effects during the finasteride session compared to the placebo session. These findings provide preliminary evidence that the risk of alcoholism associated with GABRA2 alleles may be related to differences in the subjective response to alcohol. The effects of finasteride provide indirect evidence for a mediating role of neuroactive steroids in some of the subjective effects of alcohol.

AbstractExposure to alcohol during adolescence impacts cortical and limbic brain regions undergoing maturation. In rodent models, long-term effects on behavior and neurophysiology have been described after adolescent intermittent ethanol (AIE), especially in males. We hypothesized that AIE in female rats increases conditional approach to a reward-predictive cue and corresponding neuronal activity in the orbitofrontal cortex (OFC) and nucleus accumbens (NAc). We evaluated behavior and neuronal firing after AIE (5 g/kg intragastric) or water (CON) in adult female rats. Both AIE and CON groups expressed a ST phenotype, and AIE marginally increased sign-tracking (ST) and decreased goal-tracking (GT) metrics. NAc neurons exhibited phasic firing patterns to the conditional stimulus (CS), with no differences between groups. In contrast, neuronal firing in the OFC of AIE animals was greater at CS onset and offset than in CON animals. During reward omission, OFC responses to CS offset normalized to CON levels, but enhanced OFC firing to CS onset persisted in AIE. We suggest that the enhanced OFC neural activity observed in AIE rats to the CS could contribute to behavioral inflexibility. Ultimately, AIE persistently impacts the neurocircuitry of reward-motivated behavior in female rats.

AbstractProgression of chronic infections to end‐stage diseases and poor treatment results are frequently associated with alcohol abuse. Alcohol metabolism suppresses innate and adaptive immunity leading to increased viral load and its spread. In case of hepatotropic infections, viruses accelerate alcohol‐induced hepatitis and liver fibrosis, thereby promoting end‐stage outcomes, including cirrhosis and hepatocellular carcinoma (HCC). In this review, we concentrate on several unexplored aspects of these phenomena, which illustrate the combined effects of viral/bacterial infections and alcohol in disease development. We review alcohol‐induced alterations implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis in Simian immunodeficiency virus/human immunodeficiency virus infection. Furthermore, in hepatocytes, both HIV infection and alcohol activate oxidative stress to cause lysosomal dysfunction and leakage and apoptotic cell death, thereby increasing hepatotoxicity. In addition, we discuss the mechanisms of hepatocellular carcinoma and tumor signaling in hepatitis C virus infection. Finally, we analyze studies that review and describe the immune derangements in hepatotropic viral infections focusing on the development of novel targets and strategies to restore effective immunocompetency in alcohol‐associated liver disease. In conclusion, alcohol exacerbates the pathogenesis of viral infections, contributing to a chronic course and poor outcomes, but the mechanisms behind these events are virus specific and depend on virus–alcohol interactions, which differ among the various infections.