Effects Of Chronic Alcohol Exposure Research Articles

Investigation of the Effects of Chronic Alcohol Exposure on the Spleen Using Stereological and Immunohistochemical Methods

Amaç: Alkol toksik bir ajan olup, gün geçtikçe tüketimi artmaktadır. Tüm doku ve organlar üzerinde zararlı etkileri mevcut olan alkolün, çalışmamızda dalak üzerinde oluşturduğu etkileri stereolojik ve immunohistokimyasal yöntemlerle incelemeyi amaçladık. 
 Materyal ve Metot: Çalışma için 13 adet Wistar albino cinsi erkek sıçan alındı. Rastgele Kontrol (6), Etanol (7) olarak 2 gruba ayrıldı. Etanol grubuna oral yolla su yerine %6,4 (v/v) dozunda etanol verildi. Kontrol grubuna herhangi bir uygulama yapılmadı. Uygulamalara 18 gün devam edildi. Deney sonunda anestezi altında dalak dokusu çıkarıldı. Rutin ışık mikroskopik histolojik takip prosedürü ve immunohistokimyasal boyama için doku hazırlık aşamaları uygulandı, parafine gömüldü. Akabinde 5 µm kalınlığında ilki rastgele olmak üzere her 40. kesit alınarak, ortalama 10 kesit alındı. Alınan kesitler Hematoksilen & Eosinle boyandı. Stereolojik ölçümde Cavalieri prensibinin modifiye metodu kullanıldı. Shtereom 1.5 version paket programında verilen noktalı alan cetveliyle total doku volüm oranları ölçüldü. Doku kesitlerine Kollajen Tip IV ve Fibronektin immunohistokimyasal boyama uygulandı. İmmunohistokimyasal ve stereolojik değerlendirmeler yapıldı. Sonuçlar istatistiki olarak karşılaştırıldı.
 Bulgular: Stereolojik ölçümlerde dalak total volümü ve kırmızı pulpa volümünün etanol grubunda kontrol grubuna göre artmış olduğu ve istatistiki olarak anlamlı olduğu tespit edildi (p0.05). Kollajen Tip IV ve Fibronektin’in immunuhistokimyasal değerlendirilmesinde kapsül ve trabeküller alanlarda kontrol grubuyla karşılaştırıldığında etanol grubunda tutulumunun ciddi oranda azalmış olduğu tespit edildi. 
 Sonuç: Çalışmamızın stereolojik ve immunohistokimyasal bulgularıyla alkolün immun ve hematopoetik sistemin kavşak noktasında yer alan dalak üzerindeki olumsuz etkileri ortaya konuldu.

Transcriptional Dysregulation of Cholesterol Synthesis Underlies Hyposensitivity to GABA in the Ventral Tegmental Area During Acute Alcohol Withdrawal

BackgroundThe ventral tegmental area (VTA) is a dopaminergic brain area that is critical in the development and maintenance of addiction. During withdrawal from chronic ethanol exposure, the response of VTA neurons to GABA (gamma-aminobutyric acid) is reduced through an epigenetically regulated mechanism. In the current study, a whole-genome transcriptomic approach was used to investigate the underlying molecular mechanism of GABA hyposensitivity in the VTA during withdrawal after chronic ethanol exposure. MethodsWe performed RNA sequencing of the VTA of Sprague Dawley male rats withdrawn for 24 hours from a chronic ethanol diet as well as sequencing of the VTA of control rats fed the Lieber-DeCarli diet. RNA sequencing data were analyzed using weighted gene coexpression network analysis to identify modules that contained coexpressed genes. Validation was performed with quantitative polymerase chain reaction, gas chromatography–mass spectrometry, and electrophysiological assays. ResultsPathway and network analysis of weighted gene coexpression network analysis module 1 revealed a significant downregulation of genes associated with the cholesterol synthesis pathway. Consistent with this association, VTA cholesterol levels were significantly decreased during withdrawal. Chromatin immunoprecipitation indicated a decrease in levels of acetylated H3K27 at the transcriptional control regions of these genes. Electrophysiological studies in VTA slices demonstrated that GABA hyposensitivity during withdrawal was normalized by addition of exogenous cholesterol. In addition, inhibition of cholesterol synthesis produced GABA hyposensitivity, which was reversed by adding exogenous cholesterol to VTA slices. ConclusionsThese results suggest that decreased expression of cholesterol synthesis genes may regulate GABA hyposensitivity of VTA neurons during alcohol withdrawal. Increasing cholesterol levels in the brain may be a novel avenue for therapeutic intervention to reverse detrimental effects of chronic alcohol exposure.

The effect of chronic alcohol exposure on spatial memory and BDNF–TrkB- PLCγ1 signaling in the hippocampus of male and female mice

Alcohol is a commonly used drug worldwide, and abuse of alcohol has become a serious public health problem. Alcohol consumption over time can cause cognitive deficits and memory impairment, which is thought to be associated with changes in the hippocampus. Given previously known effects of brain-derived neurotrophic factor (BDNF) in regulating synaptic plasticity and learning and memory, we investigated the effect of chronic alcohol consumption on spatial memory impairment in both sexes and changes in BDNF signaling in the hippocampus. After 4 weeks of intermittent access to 20% alcohol, memory impairment in both male and female mice was evaluated using the Morris water maze and the expression of BDNF, TrkB, phosphorylation of PLCγ1 (p-PLCγ1) and PLCγ1 in the hippocampus was examined using Western blot. As expected, females spent longer escape latencies during the training phase, and both sexes spent shorter time in the target quadrant. Furthermore, after 4 weeks 20% alcohol exposure, we found significantly decreased expression levels of BDNF in the hippocampus of female mice but increased levels in male mice. TrkB and PLCγ1 expression showed no significant change in the hippocampus of both sexes. These findings suggest that chronic alcohol exposure may induce spatial memory impairment in both sexes and opposite changes in expression of BDNF and p-PLCγ1 in the hippocampus of males and females.

Sex differences in the transcriptome of extracellular vesicles secreted by fetal neural stem cells and effects of chronic alcohol exposure

BackgroundPrenatal alcohol (ethanol) exposure (PAE) results in brain growth restriction, in part, by reprogramming self-renewal and maturation of fetal neural stem cells (NSCs) during neurogenesis. We recently showed that ethanol resulted in enrichment of both proteins and pro-maturation microRNAs in sub-200-nm-sized extracellular vesicles (EVs) secreted by fetal NSCs. Moreover, EVs secreted by ethanol-exposed NSCs exhibited diminished efficacy in controlling NSC metabolism and maturation. Here we tested the hypothesis that ethanol may also influence the packaging of RNAs into EVs from cell-of-origin NSCs.MethodsSex-specified fetal murine iso-cortical neuroepithelia from three separate pregnancies were maintained ex vivo, as neurosphere cultures to model the early neurogenic niche. EVs were isolated by ultracentrifugation from NSCs exposed to a dose range of ethanol. RNA from paired EV and cell-of-origin NSC samples was processed for ribosomal RNA-depleted RNA sequencing. Differential expression analysis and exploratory weighted gene co-expression network analysis (WGCNA) identified candidate genes and gene networks that were drivers of alterations to the transcriptome of EVs relative to cells.ResultsThe RNA content of EVs differed significantly from cell-of-origin NSCs. Biological sex contributed to unique transcriptome variance in EV samples, where > 75% of the most variant transcripts were also sex-variant in EVs but not in cell-of-origin NSCs. WGCNA analysis also identified sex-dependent enrichment of pathways, including dopamine receptor binding and ectoderm formation in female EVs and cell-substrate adhesion in male EVs, with the top significant DEGs from differential analysis of overall individual gene expressions, i.e., Arhgap15, enriched in female EVs, and Cenpa, enriched in male EVs, also serving as WCGNA hub genes of sex-biased EV WGCNA clusters. In addition to the baseline RNA content differences, ethanol exposure resulted in a significant dose-dependent change in transcript expression in both EVs and cell-of-origin NSCs that predominantly altered sex-invariant RNAs. Moreover, at the highest dose, ~ 73% of significantly altered RNAs were enriched in EVs, but depleted in NSCs.ConclusionsThe EV transcriptome is distinctly different from, and more sex-variant than, the transcriptome of cell-of-origin NSCs. Ethanol, a common teratogen, results in dose-dependent sorting of RNA transcripts from NSCs to EVs which may reprogram the EV-mediated endocrine environment during neurogenesis.

Effects of chronic alcohol exposure on motivation-based value updating.

Dysfunctional decision-making has been observed in alcohol dependence. However, the specific underlying processes disrupted have yet to be identified. Important to goal-directed decision-making is one's motivational state, which is used to update the value of actions. As ethanol dependence disrupts decision-making processes, we hypothesized that ethanol dependence could alter sensitivity to motivational state and/or value updating, thereby reducing the capability for adaptive behavior. Here we employed a sequential instrumental learning task to examine this hypothesis. In two experiments, mice underwent chronic intermittent ethanol (CIE) or air (Air) vapor exposure and repeated withdrawal procedures to induce ethanol dependence. Mice were then trained on a sequence of distal and proximal lever pressing for sucrose under either mild or more severe food restriction. Half of all Air and CIE mice then underwent a motivational shift to a less hungry state and effects of this motivational shift were evaluated across three days. First, mice were re-exposed to sucrose, and effects of food restriction state and CIE exposure on lick and consummatory behavior were examined in the absence of lever pressing. Over the next two days, mice underwent a brief non-rewarded test and then a rewarded test where the ability to retrieve and infer sucrose value to guide lever pressing was measured. In the sucrose re-exposure session, prior CIE exposure altered sucrose-seeking in mice with a history of mild but not more severe food restriction, suggesting altered motivational sensitivity. During lever press testing, CIE mice were insensitive to decreases in motivational state and did not reduce proximal lever pressing regardless of food restriction state. Mildly restricted CIE mice, but not severely restricted CIE mice, also did not reduce distal pressing to the same degree as Air mice following a downshift in motivational state. Our findings suggest that ethanol dependence may disrupt motivational processes supporting value updating that are important for decision-making.

Chronic Alcohol Exposure Alters Gene Expression and Neurodegeneration Pathways in the Brain of Adult Mice.

Chronic alcohol consumption can alter the structure of the central nervous system and disrupt cognitive function. Alcoholics are more likely to develop neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). However, the role of alcohol in promoting neurotoxicity and neurodegeneration remains unclear. In this study, we aimed at estimating the effects of chronic binge alcohol exposure on brain transcriptome and behavior changes in a chronic "Drinking in the Dark" (DID) mouse model. The adult C57BL/6J male mice were exposed to alcohol for 4 weeks. RNA-seq was applied to assess the effects of chronic alcohol exposure on transcriptome in brain. The open field test and novel object recognition test were used to assess the changes of anxiety level, locomotive function, and short-term memory induced by alcohol. RNA-seq analysis revealed that chronic alcohol exposure caused significant change in the brain transcriptome, especially in prefrontal cortex. The gene dysregulation caused by chronic alcohol exposure includes pathways related to mitochondrial energy metabolism (such as oxidative phosphorylation) and multiple neurodegenerative diseases (such as AD and PD). Furthermore, the pathway and network analyses suggest that the genes involved in mitochondrial energy metabolism, ubiquitin-proteasome system, Wnt signaling pathway, and microtubules may attribute to the neurotoxicity and neurodegeneration caused by chronic alcohol consumption. Additionally, locomotive function was also significantly impaired. This work provides gene transcriptional profile data for future research on alcohol-induced neurodegenerative diseases, especially AD and PD.

Alcohol Dependence in Rats Is Associated with Global Changes in Gene Expression in the Central Amygdala.

Alcohol dependence is associated with adverse consequences of alcohol (ethanol) use and is evident in most severe cases of alcohol use disorder (AUD). The central nucleus of the amygdala (CeA) plays a critical role in the development of alcohol dependence and escalation of alcohol consumption in dependent subjects. Molecular mechanisms underlying the CeA-driven behavioral changes are not well understood. Here, we examined the effects of alcohol on global gene expression in the CeA using a chronic intermittent ethanol (CIE) vapor model in rats and RNA sequencing (RNA-Seq). The CIE procedure resulted in robust changes in CeA gene expression during intoxication, as the number of differentially expressed genes (DEGs) was significantly greater than those expected by chance. Over-representation analysis of cell types, functional groups and molecular pathways revealed biological categories potentially important for the development of alcohol dependence in our model. Genes specific for astrocytes, myelinating oligodendrocytes, and endothelial cells were over-represented in the DEG category, suggesting that these cell types were particularly affected by the CIE procedure. The majority of the over-represented functional groups and molecular pathways were directly related to the functions of glial and endothelial cells, including extracellular matrix (ECM) organization, myelination, and the regulation of innate immune response. A coordinated regulation of several ECM metalloproteinases (e.g., Mmp2; Mmp14), their substrates (e.g., multiple collagen genes and myelin basic protein; Mbp), and a metalloproteinase inhibitor, Reck, suggests a specific mechanism for ECM re-organization in response to chronic alcohol, which may modulate neuronal activity and result in behavioral changes, such as an escalation of alcohol drinking. Our results highlight the importance of glial and endothelial cells in the effects of chronic alcohol exposure on the CeA, and demonstrate further insight into the molecular mechanisms of alcohol dependence in rats. These molecular targets may be used in future studies to develop therapeutics to treat AUD.

Chronic Exposure to Alcohol Inhibits New Myelin Generation in Adult Mouse Brain.

Chronic alcohol consumption causes cognitive impairments accompanying with white matter atrophy. Recent evidence has shown that myelin dynamics remain active and are important for brain functions in adulthood. For example, new myelin generation is required for learning and memory functions. However, it remains undetermined whether alcohol exposure can alter myelin dynamics in adulthood. In this study, we examine the effect of chronic alcohol exposure on myelin dynamics by using genetic approaches to label newly generated myelin (NG2-CreERt; mT/mG). Our results indicated that alcohol exposure (either 5% or 10% in drinking water) for 3 weeks remarkably reduced mGFP + /NG2- new myelin and mGFP + /CC1 + new oligodendrocytes in the prefrontal cortex and corpus callosum of 6-month-old NG2-CreERt; mT/mG mice as compared to controls without changing the mGFP + /NG2 + oligodendrocyte precursor cells (OPCs) density, suggesting that alcohol exposure may inhibit oligodendrocyte differentiation. In support with these findings, the alcohol exposure did not significantly alter apoptotic cell number or overall MBP expression in the brains. Further, the alcohol exposure decreased the histone deacetylase1 (HDAC1) expression in mGFP + /NG2 + OPCs, implying epigenetic mechanisms were involved in the arrested OPC differentiation. Together, our results indicate that chronic exposure to alcohol can inhibit myelinogenesis in the adult mouse brain and that may contribute to alcohol-related cognitive impairments.

Effect of chronic alcohol exposure on gut vitamin B7 uptake: involvement of epigenetic mechanisms and effect of alcohol metabolites

Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.

Elevated thalamic glutamate levels and reduced water diffusivity in alcohol use disorder: Association with impulsivity

Alcohol induces neuroinflammation but its role in cognitive impairment and impulsivity in alcohol use disorder (AUD) has been poorly investigated. We used proton magnetic resonance spectroscopy to measure brain glutamate (Glu) levels and diffusion-weighted imaging to measure functional anisotropy (FA) in the thalamus and ventral anterior cingulate cortex (vACC) in 15 recently detoxified patients with AUD and 14 matched controls. Compared to controls, AUD patients showed higher Glu levels (p = 0.04) and lower FA in the thalamus (p = 0.04) but not in the vACC. In AUD, thalamic Glu levels (r = 0.62, p = 0.019) and FA (r=-0.55, p = 0.034) were associated with severity of drinking (drinks/week). Compared to controls, AUD patients showed higher scores on Conners’ Adult ADHD Rating Scale for impulsivity (p = 0.03), which correlated with glutamate levels in the thalamus (r = 0.58, p = 0.03) and vACC (r = 0.55, p = 0.036). In a second cohort of AUD patients (n = 32), Glu in dorsal ACC (dACC) also correlated with Barrett Impulsiveness Scale total score (r = 0.43, p = 0.014). We interpret the elevated thalamic Glu levels and the parallel reduction in FA in AUD—which correlated with drinking severity—as possible evidence of neurotoxicity from neuroinflammation. The association of Glu with impulsivity suggests that neurotoxic effects of chronic alcohol exposure in the thalamus and dACC may contribute to impulsivity.

Selective Deficits in Contingency-Driven Ethanol Seeking Following Chronic Ethanol Exposure in Male Mice.

People with alcohol use disorders exhibit an overreliance on habitual response strategies which may result from a history of chronic alcohol exposure. Although habits are defined by behavior that persists despite changes in outcome value and in action-outcome relationships, most research investigating the effects of ethanol exposure on habits has focused only on outcome devaluation. A clear understanding of the effects of chronic alcohol exposure on the ability to flexibly update behavior may provide insight into the behavioral deficits that characterize alcohol use disorders. To dissociate the effects of chronic intermittent ethanol (CIE) exposure on contingency-mediated sucrose versus ethanol seeking, adult male C57Bl/6J mice were assigned to 2 separate experiments. In the first experiment, mice were trained to self-administer ethanol prior to 2 cycles of interleaved CIE exposure by vapor inhalation. In a second experiment, mice were trained to self-administer sucrose and ethanol in separate training sessions prior to 4 cycles of interleaved CIE. The use of contingencies to mediate reward seeking was assessed using a contingency degradation paradigm. In mice trained to self-administer only ethanol, 2weeks of CIE resulted in escalated self-administration. At this time point, CIE-exposed mice, but not air-exposed controls, exhibited ethanol seeking that was insensitive to changes in action-outcome contingency, consistent with habitual ethanol seeking. In mice trained to self-administer ethanol and sucrose rewards in sequential sessions, no escalation in self-administration across 4weeks of CIE was observed. Under these conditions, neither Air- nor CIE-exposed mice reduced ethanol seeking in response to contingency degradation. In contrast, sucrose seeking remained goal-directed. Our results suggest that chronic ethanol exposure impairs contingency-driven ethanol seeking more readily than sucrose-seeking behavior. Further, these findings indicate that the transition from contingency-mediated ethanol seeking occurs more rapidly than for sucrose seeking under similar ethanol exposure conditions.

The Effects of PK11195 and Protoporphyrin IX Can Modulate Chronic Alcohol Intoxication in Rat Liver Mitochondria under the Opening of the Mitochondrial Permeability Transition Pore.

Decades of active research have shown that mitochondrial dysfunction, the associated oxidative stress, impaired anti-stress defense mechanisms, and the activation of the proapoptotic signaling pathways underlie pathological changes in organs and tissues. Pathologies caused by alcohol primarily affect the liver. Alcohol abuse is the cause of many liver diseases, such as steatosis, alcoholic steatohepatitis, fibrosis, cirrhosis, and, potentially, hepatocellular cancer. In this study, the effect of chronic alcohol exposure on rat liver mitochondria was investigated. We observed an ethanol-induced increase in sensitivity to calcium, changes in the level of protein kinase Akt and GSK-3β phosphorylation, an induction of the mitochondrial permeability transition pore (mPTP), and strong alterations in the expression of mPTP regulators. Moreover, we also showed an enhanced effect of PK11195 and PPIX, on the parameters of the mPTP opening in rat liver mitochondria (RLM) isolated from ethanol-treated rats compared to the RLM from control rats. We suggest that the results of this study could help elucidate the mechanisms of chronic ethanol action on the mitochondria and contribute to the development of new therapeutic strategies for treating the effects of ethanol-related diseases.

Involvement of transcriptional/epigenetics mechanisms in mediating the inhibitory effect of chronic alcohol exposure, and effect of alcohol metabolites on intestinal uptake of biotin.

Biotin is indispensable for normal cellular metabolism, proliferation and survival. The vitamin acts as a co‐factor for multiple carboxylases that play critical roles in fatty acid, glucose and amino acid metabolism. Emerging evidence has also shown important roles for biotin in immune function and in gene expression, as well as in the maintenance of intestinal integrity and homeostasis. Humans and other mammals cannot synthesize biotin, and thus, must obtain the vitamin from exogenous sources (diet and the gut microbiota) via absorption in the gut. We had previously observed that chronic alcohol feeding/exposure leads to a significant inhibition in small intestinal and colonic biotin uptake (Am J Physiol Gastrointest Liver Physiol 300: G494–G501, 2011). The aim of this study was to elucidate the role of transcriptional/epigenetic factors in mediating the inhibitory effect of chronic alcohol on intestinal uptake of biotin, and to study the effect of alcohol metabolites on biotin uptake in the large intestine. Our results showed that the inhibition in biotin uptake by intestinal epithelial Caco‐2 cells upon chronic exposure to alcohol is associated with a decrease in the expression of KLF‐4, a key transcription factor involved in the regulation of SLC5A6. We also observed (using Chip‐qPCR) that exposure of Caco‐2 cells to alcohol to lead to decrease in the euchromatin histone markers (activators‐H3K4me3 and H3K9Ac) and an increase in the heterochromatin histone marker (repressor – H3K27me3) at the SLC5A6 P1 promoter. However, DNA methylation status appeared to be unaltered in these cells upon alcohol exposure. We confirmed the above effects of alcohol on histone modifications in vivo using small intestinal mucosa from mice chronically fed alcohol. We also examined the effect of the alcohol metabolites ethyl oleate (3 mM), ethyl palmitate (3 mM) and acetaldehyde (500 μM) on biotin uptake by human colonic epithelial NCM460 cells and observed a significant inhibition in carrier‐mediated biotin uptake in these cells. Studies are in progress to delineate the exact molecular mechanism(s) involved in this inhibitory effect. This study show, for the first time, that exposure of small intestinal epithelial cells to alcohol alters the epigenetic profile at the SLC5A6 promoters and the expression of an important nuclear factor that drive promoter activity, and that these changes contribute to the inhibition in biotin uptake. Further, the results show that relevant alcohol metabolites inhibit biotin uptake by colonic epithelial cells.Support or Funding InformationDVA and the NIH (DK‐56061, DK58057, and AA 018071

Hepatic SIRT3 Upregulation in Response to Chronic Alcohol Consumption Contributes to Alcoholic Liver Disease in Mice.

BackgroundAlcoholic liver disease (ALD) is a type of chronic liver disease caused by chronic ethanol overconsumption. The pathogenesis of ALD is complex and there is no effective clinical treatment thus far. SIRT3 is an NAD+-dependent deacetylase primarily located inside mitochondria, and reports on the effect of chronic alcohol exposure on liver SIRT3 expression are scarce. This study aims to investigate the effect of chronic alcohol consumption on hepatic SIRT3 expression and its role in alcoholic-induced liver injury.MethodsUsing the Lieber-DeCarli mouse model of ALD, we analyzed the regulation of SIRT3 and the effect of liver-specific knocking-down of SIRT3 on alcohol-induced liver injury. HepG2 and AML12 hepatocytes were employed to detect the biological function of SIRT3 on alcohol-induced hepatic cytotoxicity and its potential mechanism.ResultsChronic alcohol exposure led to hepatic SIRT3 upregulation and liver-specific SIRT3 knockdown alleviated alcoholic feeding-induced liver injury and lipid accumulation, which is associated with improved autophagy induction. In addition, autophagy induction contributed to the cytoprotective effect of SIRT3 knockdown on ethanol-induced hepatocyte cell death.ConclusionIn summary, our data suggest that hepatic SIRT3 upregulation in response to chronic alcohol exposure and liver-specific SIRT3 knockdown, induced autophagy activation further alleviating alcoholic-induced liver injury, which represents a novel mechanism in this process.

Glial gene networks associated with alcohol dependence

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.

Chronic alcohol causes alteration of lipidome profiling in brain

Much efforts have been tried to clarify the molecular mechanism of alcohol-induced brain damage from the perspective of genome and protein; however, the effect of chronic alcohol exposure on global lipid profiling of brain is unclear. In the present study, by using Q-TOF/MS-based lipidomics approach, we investigated the comprehensive lipidome profiling of brain from the rats orally administrated with alcohol daily, continuously for one year. Through systematically analysis of all lipids in prefrontal cortex (PFC) and striatum region, we found that long-term alcohol exposure profoundly modified brain lipidome profiling. Notably, three kinds of lipid classes, glycerophospholipid (GP), glycerolipid (GL) and fatty acyls (FA), were significantly increased in these two brain regions. Interestingly, most of the modified lipids were involved in synthetic pathways of endoplasmic reticulum (ER), which may result in ER stress-related metabolic disruption. Moreover, alcohol-modified lipid species displayed long length of carbon chain with high degree of unsaturation. Taken together, our results firstly present that chronic alcohol exposure markedly modifies brain lipidomic profiling, which may activate ER stress and eventually result in neurotoxicity. These findings provide a new insight into the mechanism of alcohol-related brain damage.

Effects of Alcohol on Inhibitory Avoidance Learning in Zebrafish (Danio rerio).

The zebrafish (Danio rerio) can be used in studies addressing the effects of drugs on learning, memory, and anxiety. In the present study, we investigated the effect of different alcohol treatments (chronic and acute) on the learning and anxiety response of zebrafish in an inhibitory avoidance paradigm. Zebrafish were initially exposed to different alcohol treatments and submitted to an inhibitory avoidance protocol, where an electroshock was applied to the fish as they swam from the white to the black side of a shuttle box tank (naturally preferred environment of zebrafish). Animals from the control and 0.5% acute alcohol groups exhibited high latency to enter the black side of the tank after the first exposure to electroshock, in addition to higher freezing and a shorter distance from the bottom of the tank, suggesting acute alcohol exposure did not affect aversive learning in zebrafish. However, chronic exposure and alcohol withdrawal impaired the fish's capacity to properly respond to the aversive stimulus. Overall, our results show the harmful effects of chronic alcohol exposure, both continued intake and its cessation, but avoidance behavior persisted and anxiety increased following acute alcohol exposure.

Effect of alcohol use disorder on oxytocin peptide and receptor mRNA expression in human brain: A post-mortem case-control study

Animal and human evidence supports a role for oxytocin in alcohol-seeking behaviors. There is interest, therefore, in targeting the oxytocin pathway as a new pharmacologic approach to treat alcohol use disorder. To this end, it is important to understand the effect of alcohol use disorder on endogenous oxytocin in brain regions that are relevant for the initiation and maintenance of alcohol use disorder. We examined human post-mortem brain tissue from males with alcohol use disorder (n=11) compared to nonalcohol dependent male controls (n=16). We a priori targeted five brain regions that in rodent studies, are projection areas for oxytocin neurons: nucleus accumbens, amygdala, hippocampus, ventral tegmental area and prefrontal cortex. Fold change in mRNA levels of oxytocin peptide and receptor were measured in each of the brain regions studied. Fold change for oxytocin peptide mRNA was significantly elevated in the prefrontal cortex of subjects with alcohol use disorder compared to controls (uncorrected p=0.0001; FDR-corrected p=0.001). For the entire sample of 27 subjects, there was a significant positive correlation between the fold change in oxytocin peptide mRNA in the prefrontal cortex and both daily alcohol intake (r2=0.38; p=0.002) and drinks per week (r2=0.24; p=0.02). Results are discussed in light of the previous animal and human literature on changes in the endogenous oxytocin system as an effect of chronic alcohol exposure.

Effects of chronic alcohol exposure on ischemia\u2013reperfusion-induced acute kidney injury in mice: the role of \u03b2-arrestin 2 and glycogen synthase kinase 3

Little is known about the effects of chronic alcohol intake on the outcome of acute kidney injury (AKI). Hence, we examined the effects of chronic alcohol intake on the development of renal fibrosis following AKI in an animal model of bilateral renal ischemia–reperfusion (IR) injury. We first found that chronic alcohol exposure exacerbated bilateral IR-induced renal fibrosis and renal function impairment. This phenomenon was associated with increased bilateral IR-induced extracellular matrix deposition and an increased myofibroblast population as well as increased bilateral IR-induced expression of fibrosis-related genes in the kidneys. To explore the mechanisms underlying this phenomenon, we showed that chronic alcohol exposure enhanced β-arrestin 2 (Arrb2) expression and Akt and glycogen synthase kinase-3 (GSK3)β activation in the kidneys. Importantly, pharmacological GSK3 inhibition alleviated bilateral IR-induced renal fibrosis and renal function impairment. Furthermore, we demonstrated that Arrb2−/− mice exhibited resistance to IR-induced renal fibrosis and renal function impairment following chronic alcohol exposure, and these effects were associated with attenuated GSK3β activation in the kidneys. Taken together, our results suggest that chronic alcohol exposure may potentiate AKI via β-arrestin 2/Akt/GSK3β-mediated signaling in the kidney.

Cellular GABAergic Neuroactive Steroid (3α,5α)-3-Hydroxy-Pregnan-20-One (3α,5α-THP) Immunostaining Levels Are Increased in the Ventral Tegmental Area of Human Alcohol Use Disorder Patients: A Postmortem Study.

The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP; allopregnanolone) enhances GABAergic activity and produces subjective effects similar to ethanol (EtOH). The effect of chronic alcohol exposure on 3α,5α-THP concentrations has been studied in mouse, rat, and monkey limbic brain areas. Chronic EtOH exposure produced divergent brain region and cell-specific changes in 3α,5α-THP concentrations in animal studies. However,3α,5α-THP levels in similar human brain regions have never been examined in individuals diagnosed with alcohol use disorder (AUD). Therefore, we used immunohistochemistry (IHC) to examine 3α,5α-THP levels in the ventral tegmental area (VTA), substantia nigra pars medialis (SNM), and amygdala of human postmortem brains of patients diagnosed with AUD compared with social drinkers. The effects of sex and liver disease on 3α,5α-THP concentrations were examined in the aforementioned brain regions. Human postmortem brains of AUD patients and age-matched controls were obtained from the New South Wales Brain Tissue Resource Center. IHC was performed using anti-3α,5α-THP antibody on formalin-fixed paraffin-embedded brain sections to detect cellular 3α,5α-THP levels. Immunoreactivity was analyzed by pixel density/mm2 for the comparison between AUD patients and controls. 3α,5α-THP immunoreactivity was increased by 23.2±9% in the VTA of AUD patients compared with age-matched controls (p=0.014). Moreover, a 29.6±10% increase in 3α,5α-THP immunoreactivity was observed in the SNM of male AUD patients compared with male controls (p<0.01), but not in female subjects. 3α,5α-THP immunoreactivity in the VTA and SNM regions did not differ between noncirrhotic and cirrhotic AUD patients. A sex difference in 3α,5α-THP immunoreactivity (female 51±18% greater than male) was observed among control subjects in the SNM, but no other brain region. 3α,5α-THP immunoreactivity in the basolateral amygdala and lateral amygdala was negatively correlated with the length of the tissue fixation time as well as the age of the subjects, precluding assessment of the effect of AUD. Cellular 3α,5α-THP levels in VTA are increased in human AUD patients, an effect that is likely independent of sex and liver disease. The differences between animal models and human studies should be factored into the interpretation of the physiological significance of elevated 3α,5α-THP levels in humans.