Excessive Ethanol Consumption Research Articles

Role of Sigma Receptors in Alcohol Addiction.

Pharmacological treatments for alcohol use disorder (AUD) are few in number and often ineffective, despite the significant research carried out so far to better comprehend the neurochemical underpinnings of the disease. Hence, research has been directed towards the discovery of novel therapeutic targets for the treatment of AUD. In the last decade, the sigma receptor system has been proposed as a potential mediator of alcohol reward and reinforcement. Preclinical studies have shown that the motivational effects of alcohol and excessive ethanol consumption involve the recruitment of the sigma receptor system. Furthermore, sigma receptor antagonism has been shown to be sufficient to inhibit many behaviors related to AUDs. This paper will review the most current evidence in support of this receptor system as a potential target for the development of pharmacological agents for the treatment of alcohol addiction.

Lipophagy and Alcohol-Induced Fatty Liver.

This review describes the influence of ethanol consumption on hepatic lipophagy, a selective form of autophagy during which fat-storing organelles known as lipid droplets (LDs) are degraded in lysosomes. During classical autophagy, also known as macroautophagy, all forms of macromolecules and organelles are sequestered in autophagosomes, which, with their cargo, fuse with lysosomes, forming autolysosomes in which the cargo is degraded. It is well established that excessive drinking accelerates intrahepatic lipid biosynthesis, enhances uptake of fatty acids by the liver from the plasma and impairs hepatic secretion of lipoproteins. All the latter contribute to alcohol-induced fatty liver (steatosis). Here, our principal focus is on lipid catabolism, specifically the impact of excessive ethanol consumption on lipophagy, which significantly influences the pathogenesis alcohol-induced steatosis. We review findings, which demonstrate that chronic ethanol consumption retards lipophagy, thereby exacerbating steatosis. This is important for two reasons: (1) Unlike adipose tissue, the liver is considered a fat-burning, not a fat-storing organ. Thus, under normal conditions, lipophagy in hepatocytes actively prevents lipid droplet accumulation, thereby maintaining lipostasis; (2) Chronic alcohol consumption subverts this fat-burning function by slowing lipophagy while accelerating lipogenesis, both contributing to fatty liver. Steatosis was formerly regarded as a benign consequence of heavy drinking. It is now recognized as the “first hit” in the spectrum of alcohol-induced pathologies that, with continued drinking, progresses to more advanced liver disease, liver failure, and/or liver cancer. Complete lipid droplet breakdown requires that LDs be digested to release their high-energy cargo, consisting principally of cholesteryl esters and triacylglycerols (triglycerides). These subsequently undergo lipolysis, yielding free fatty acids that are oxidized in mitochondria to generate energy. Our review will describe recent findings on the role of lipophagy in LD catabolism, how continuous heavy alcohol consumption affects this process, and the putative mechanism(s) by which this occurs.

Chronic alcohol exposure alters circulating insulin and ghrelin levels: role of ghrelin in hepatic steatosis.

Fatty liver is the earliest response of the liver to excessive ethanol consumption. Central in the development of alcoholic steatosis is increased mobilization of nonesterified free fatty acids (NEFAs) to the liver from the adipose tissue. In this study, we hypothesized that ethanol-induced increase in ghrelin by impairing insulin secretion, could be responsible for the altered lipid metabolism observed in adipose and liver tissue. Male Wistar rats were fed for 5-8 wk with control or ethanol Lieber-DeCarli diet, followed by biochemical analyses in serum and liver tissues. In addition, in vitro studies were conducted on pancreatic islets isolated from experimental rats. We found that ethanol increased serum ghrelin and decreased serum insulin levels in both fed and fasting conditions. These results were corroborated by our observations of a significant accumulation of insulin in pancreatic islets of ethanol-fed rats, indicating that its secretion was impaired. Furthermore, ethanol-induced reduction in circulating insulin was associated with lower adipose weight and increased NEFA levels observed in these rats. Additionally, we found that increased concentration of serum ghrelin was due to increased synthesis and maturation in the stomach of the ethanol-fed rats. We also report that in addition to its effect on the pancreas, ghrelin can also directly act on hepatocytes via the ghrelin receptors and promote fat accumulation. In conclusion, alcohol-induced elevation of circulating ghrelin levels impairs insulin secretion. Consequently, reduced circulating insulin levels likely contribute to increased free fatty acid mobilization from adipose tissue to liver, thereby contributing to hepatic steatosis. NEW & NOTEWORTHY Our studies are the first to report that ethanol-induced increases in ghrelin contribute to impaired insulin secretion, which results in the altered lipid metabolism observed in adipose and liver tissue in the setting of alcoholic fatty liver disease.

Regional Differences and Similarities in the Brain Transcriptome for Mice Selected for Ethanol Preference From HS-CC Founders.

The high genetic complexity found in heterogeneous stock (HS-CC) mice, together with selective breeding, can be used to detect new pathways and mechanisms associated with ethanol preference and excessive ethanol consumption. We predicted that these pathways would provide new targets for therapeutic manipulation. Previously (Colville et al., 2017), we observed that preference selection strongly affected the accumbens shell (SH) genes associated with synaptic function and in particular genes associated with synaptic tethering. Here we expand our analyses to include substantially larger sample sizes and samples from two additional components of the “addiction circuit,” the central nucleus of the amygdala (CeA) and the prelimbic cortex (PL). At the level of differential expression (DE), the majority of affected genes are region-specific; only in the CeA did the DE genes show a significant enrichment in GO annotation categories, e.g., neuron part. In all three brain regions the differentially variable genes were significantly enriched in a single network module characterized by genes associated with cell-to-cell signaling. The data point to glutamate plasticity as being a key feature of selection for ethanol preference. In this context the expression of Dlg2 which encodes for PSD-93 appears to have a key role. It was also observed that the expression of the clustered protocadherins was strongly associated with preference selection.

Tributyrin Supplementation Protects Immune Responses and Vasculature and Reduces Oxidative Stress in the Proximal Colon of Mice Exposed to Chronic-Binge Ethanol Feeding.

Excessive ethanol consumption causes adverse effects and contributes to organ dysfunction. Ethanol metabolism triggers oxidative stress, altered immune function, and gut dysbiosis. The gut microbiome is known to contribute to the maintenance of intestinal homeostasis, and disturbances are associated with pathology. A consequence of gut dysbiosis is also alterations in its metabolic and fermentation byproducts. The gut microbiota ferments undigested dietary polysaccharides to yield short-chain fatty acids, predominantly acetate, propionate, and butyrate. Butyrate has many biological mechanisms of action including anti-inflammatory and immunoprotective effects, and its depletion is associated with intestinal injury. We previously showed that butyrate protects gut-liver injury during ethanol exposure. While the intestine is the largest immune organ in the body, little is known regarding the effects of ethanol on intestinal immune function. This work is aimed at investigating the effects of butyrate supplementation, in the form of the structured triglyceride tributyrin, on intestinal innate immune responses and oxidative stress following chronic-binge ethanol exposure in mice. Our work suggests that tributyrin supplementation preserved immune responses and reduced oxidative stress in the proximal colon during chronic-binge ethanol exposure. Our results also indicate a possible involvement of tributyrin in maintaining the integrity of intestinal villi vasculature disrupted by chronic-binge ethanol exposure.

Chlorogenic acid ameliorates alcohol-induced liver injuries through scavenging reactive oxygen species

The key role of oxidative stress in alcoholic liver disease (ALD) has been established by the large body of evidence from previous studies. Excessive consumption of ethanol induces the production of a variety of reactive oxygen species (ROS) in the liver, such as superoxide, H2O2, and hydroxyl radical. These products activate oxidant-sensitive signaling cascades and modulators of apoptosis. Because ROS accumulation is closely related to ALD, a number of studies have investigated the benefits of antioxidants. Recent studies demonstrated that polyphenol chlorogenic acid (CGA) has antioxidant properties and health benefits, such as reduction of relative risk of cardiovascular diseases and hepatoprotective effects against acetaminophen toxicity. However, the protective effects of CGA against ALD have not been studied in detail. We hypothesize that CGA plays a role in preventing ALD through its antioxidant properties. In this study, we investigated the protective effects of CGA against liver injuries in vivo. Reduced alcohol-induced-steatosis, apoptotic cell death, and fibrosis due to reduced levels of oxidative stress were observed. These findings suggest that CGA treatment can be an effective approach to attenuate ALD through the suppression of oxidative stress.

New, previously unreported correlations between latent Toxoplasma gondii infection and excessive ethanol consumption

A number of world literature reports indicate that a latent Toxoplasma gondii infection leads to development of central nervous system disorders, which in turn may lead to altered behavior in the affected individuals. T. gondii infection has been observed to play the greatest role in drivers, suicides, and psychiatric patients. Studies conducted for this manuscript involve a different, never before really reported correlation between latent T. gondii infection and ethanol abuse. A total of 538 decedents with a known cause of death were included in the study. These individuals were divided into three groups: the risky behavior group, inconclusively risky behavior group, and control group. The criterion for this division was the likely effect of the individual’s behavior on the mechanism and cause of his/her death. The material used for analyses were blood samples collected during routine medico-legal examinations in these cases. The blood samples were used to measure anti-T. gondii IgG antibodies with an enzyme-linked immunosorbent assay (ELISA). Moreover, the following data were recorded for each decedent: sex, age, circumstances of death, cause of death, time from death to autopsy, and (if provided) substance abuse status (alcohol, illicit drugs). In those cases where blood alcohol level or toxicology tests were requested by the Prosecutor’s Office, their results were also included in our analysis. Test results demonstrated a strong correlation between latent T. gondii infection and engaging in risky behaviors leading to death. Moreover, analyses demonstrated a positive correlation between the presence of anti-T. gondii IgG antibodies and psychoactive substance (especially ethanol) abuse, however, the causal relationship remains unclear. Due to the fact that alcohol abuse constitutes a significant social problem, searching for eliminable risk factors for addiction is extremely important. Our analyses provided new important information on the possible effects of latent T. gondii infection in humans.

Nucleus Accumbens MC4-R Stimulation Reduces Food and Ethanol Intake in Adult Rats Regardless of Binge-Like Ethanol Exposure during Adolescence.

The melanocortin (MC) system regulates feeding and ethanol consumption. Recent evidence shows that melanocortin 4 receptor (MC4-R) stimulation within the nucleus accumbens (NAc) elicits anorectic responses and reduces ethanol consumption and ethanol palatability in adult rats. Ethanol exposure during adolescence causes long-lasting changes in neural pathways critically involved in neurobehavioral responses to ethanol. In this regard, binge-like ethanol exposure during adolescence reduces basal alpha-melanocyte-stimulating hormone (α-MSH) and alters the levels of agouti-related peptide (AgRP) in hypothalamic and limbic areas. Given the protective role of MC against excessive ethanol consumption, disturbances in the MC system induced by binge-like ethanol exposure during adolescence might contribute to excessive ethanol consumption during adulthood. In the present study, we evaluated whether binge-like ethanol exposure during adolescence leads to elevated ethanol intake and/or eating disturbance during adulthood. Toward that aim, Sprague-Dawley rats were treated with ethanol (3 g/kg i.p.; BEP group) or saline (SP group) for 14 days (PND 25 to PND 38). On PND73, all the groups were given access to 20% ethanol on an intermittent schedule. Our results showed that adult rats given intermittent access (IAE) to 20% ethanol achieved high spontaneous ethanol intake that was not significantly enhanced by binge-like ethanol pretreatment during adolescence. However, BEP group exhibited an increase in food intake without a parallel increase in body weight (BW) relative to SP group suggesting caloric efficiency disturbance. Additionally, we evaluated whether binge-like ethanol exposure during adolescence alters the expected reduction in feeding and ethanol consumption following NAc shell administration of a selective MC4-R agonist in adult rats showing high rates of ethanol consumption. For that, animals in each pretreatment condition (SP and BEP) were divided into three subgroups and given bilateral NAc infusions of the selective MC4-R agonist cyclo(NH-CH2-CH2-CO-His-D-Phe-Arg-Trp-Glu)-NH2 (0, 0.75 or 1.5 μg). Results revealed that MC4-R stimulation within the NAc reduced feeding and ethanol intake in high ethanol-drinking adult rats, regardless of previous binge-like ethanol exposure during adolescence, which adds new evidence regarding the dual ability of MC compounds to control excessive ethanol and food intake.

Brain transcriptional changes in the mouse and macaque associated with excessive ethanol consumption

Brain transcriptional changes in the mouse and macaque associated with excessive ethanol consumption

Alcohol drinking induced alterations in dynorphin signaling in the extended amygdala

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines.We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression.We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter.Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.

Alignment of the transcriptome with individual variation in animals selectively bred for High Drinking-In-the-Dark (HDID).

Among animals at risk for excessive ethanol consumption such as the HDID selected mouse lines, there is considerable individual variation in the amount of ethanol consumed and the associated blood ethanol concentrations (BECs). For the HDID lines, this variation occurs even though the residual genetic variation associated with the DID phenotype has been largely exhausted and thus is most likely associated with epigenetic factors. Here we focus on the question of whether the genes associated with individual variation in HDID-1 mice are different from those associated with selection (risk) (Iancu etal., 2013). Thirty-three HDID-1 mice were phenotyped for their BECs at the end of a standard DID trial, were sacrificed 3 weeks later, and RNA-Seq was used to analyze the striatal transcriptome. The data obtained illustrate that there is considerable overlap of the risk and variation gene sets, both focused on the fine-tuning of synaptic plasticity.

Oxytocin Reduces Ethanol Self-Administration in Mice.

Excessive ethanol (EtOH) consumption remains an important health concern and effective treatments are lacking. The central oxytocin system has emerged as a potentially important therapeutic target for alcohol and drug addiction. These studies tested the hypothesis that oxytocin reduces EtOH consumption. Male C57BL/6J mice were given access to EtOH (20% v/v) using a model of binge-like drinking ("drinking in the dark") that also included the use of lickometer circuits to evaluate the temporal pattern of intake as well as 2-bottle choice drinking in the home cage. In addition, EtOH (12% v/v) and sucrose (5% w/v) self-administration on fixed- and progressive-ratio schedules were also evaluated. A wide range of systemically administered oxytocin doses were tested (0 to 10mg/kg) in these models. Oxytocin (0, 0.3, 1, 3, or 10mg/kg) dose dependently reduced EtOH consumption (maximal 45% reduction) in the binge drinking model, with lower effective doses having minimal effects on general locomotor activity. Oxytocin's effect was blocked by pretreatment with an oxytocin receptor antagonist, and the pattern of contacts (licks) at the EtOH bottle suggested a reduction in motivation to drink EtOH. Oxytocin decreased 2-bottle choice drinking without altering general fluid intake. Oxytocin also reduced operant responding for EtOH and sucrose in a dose-related manner. However, oxytocin decreased responding and motivation (breakpoint values) for EtOH at doses that did not alter responding for sucrose. These results indicate that oxytocin reduces EtOH consumption in different models of self-administration. The effects are not likely due to a general sedative effect of the neuropeptide. Further, oxytocin reduces motivation for EtOH at doses that do not alter responding for a natural reward (sucrose). While some evidence supports a role for oxytocin receptors in mediating these effects, additional studies are needed to further elucidate underlying mechanisms. Nevertheless, these results support the therapeutic potential of oxytocin as a treatment for alcohol use disorder.

Delta-opioid receptor antagonism leads to excessive ethanol consumption in mice with enhanced activity of the endogenous opioid system

The opioid system modulates the central reinforcing effects of ethanol and participates in the etiology of addiction. However, the pharmacotherapy of ethanol dependence targeted on the opioid system is little effective and varies due to individual patients' sensitivity. In the present study, we used two mouse lines with high (HA) and low (LA) activity of the endogenous opioid system to analyze the effect of opioid receptor blockade on ethanol drinking behavior. We found that LA and HA lines characterized by divergent magnitudes of swim stress-induced analgesia also differ in ethanol intake and preference. Downregulation of the opioid system in LA mice was associated with increased ethanol consumption. Treatment with a non-selective opioid receptor antagonist (naloxone) had no effect on ethanol intake in this line. Surprisingly, in HA mice, the blockage of opioid receptors led to excessive ethanol consumption. Moreover, naloxone selectively induced high levels of anxiety- and depressive-like behaviors in HA mice which was attenuated by ethanol. With the use of specific opioid receptor antagonists we showed that the naloxone-induced increase in ethanol drinking in HA mice is mediated mainly by δ and to a lower extent by μ opioid receptors. The effect of δ-opioid receptor antagonism was abolished in HA mice carrying a C320T transition in the δ-opioid receptor gene (EU446125.1), which impairs this receptor's function. Our results indicate that high activity of the opioid system plays a protective role against ethanol dependence. Therefore, its blockage with opioid receptor antagonists may lead to a profound increase in ethanol consumption.

Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence.

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.

Ethanol enhances thymocyte apoptosis and autophagy in macrophages of rat thymi.

Tingible body macrophages (TBMs) play essential roles in the phagocytosis of apoptotic lymphocytes, specifically under exposure to various stressors. Although excessive ethanol consumption may enhance thymocyte apoptosis, reports investigating the autophagic response of the thymus to ethanol toxicity are still lacking. We investigated apoptosis and autophagy in thymi of an animal model of binge ethanol exposure. Adult male Wistar rats were injected intraperitoneally either with 5 g/kg ethanol or phosphate buffer saline (for the control group) and sacrificed 0, 3, 6 and 24 hours after injection. Light and transmission electron microscopy (TEM) studies revealed enhanced formation of TBMs phagocytosing many apoptotic thymocytes in the thymic cortex of the ethanol-treated rats (ETRs), and this formation was particularly marked at 24 h. The macrophages showed signs of activation under TEM and immunofluorescence double labeling with RM4 (a macrophage marker) and iNOS. Additionally, in comparison to the control group, autophagy was enhanced in ETR thymic TBMs as evidenced ultrastructurally by accumulation of autophagic vacuoles, immunohistochemical increases in LC3 puncta, Western blot analysis of the latter protein, and colocalization of LC3 and RM4 in immunofluorescence double labeling. Immunoelectron microscopy also revealed LC3-labeled autophagic vacuoles and apoptotic cell phagosomes in ETR TBMs, suggesting the possibility of LC3-related phagocytosis. This was confirmed by enhanced colocalization of LC3 with lysosomal cathepsins in double labeling. These results indicate that enhanced autophagy in ETR thymic TBMs is not only a cytoprotective mechanism but could also be involved in the clearance of apoptotic thymocytes, thus preventing autoimmune reactions and suppressing inflammatory response.

332 - Ethanol Withdrawal Induces Oxidative Damage and Hypertension: Role of AT1 Receptor Activation

Ethanol withdrawal (EW) is defined as the abrupt termination of excessive long-term ethanol consumption. EW causes oxidative damage and hypertension. We used a model of EW involving abrupt cessation of ethanol consumption (3-9% ethanol v/v for 21 days) for 48 hours. During the 48h period of EW, male Wistar rats were treated with losartan (LST - 10 mg/kg/daily, gavage) to investigate the role of angiotensin II on the systemic and tissue oxidative stress induced by EW. Oxidative markers (superoxide anion - O2–; thiobarbituric acid reactive substances – TBARS; hydrogen peroxide - H2O2), nitrate/nitrite (NOx), and antioxidant activity (superoxide dismutase – SOD; glutathione - GSH) were evaluated in plasma and aorta. Systolic blood pressure (SBP) was measured by plethysmography. Two-way ANOVA followed by Bonferroni test was used to analyze the results. Ethics Committee protocol (#11.1.1432.53.5). EW induced an imbalance in the production of reactive oxygen species (ROS) in the aortic tissue, represented by an increase in O2– generation and TBARS levels, and a decrease in H2O2 levels. Qualitative analysis in situ using the dyes DHE and DCFH-DA corroborated these findings. LST prevented these responses. In plasma, EW increased TBARS production and treatment with LST prevented this response. EW induced progression in oxidative damage and decreased the levels of NOx in plasma and aorta, and treatment with LST prevented these responses. No difference was found in plasma or aortic SOD activity. In plasma, but not aorta, EW increased GSH levels and LST did not prevent this response. Finally, we observed that LST prevented EW-induced increase in SBP. These findings show that angiotensin II plays a role on EW-induced oxidative damage and hypertension.

Evaluating the effects of refined carbohydrate and fat diets with acute ethanol consumption using a mouse model of alcoholic liver injury

Alcoholism is a multifactorial and complex disorder responsible for 5.9% of deaths worldwide. Excessive consumption of ethanol (Et-OH) induces alcoholic liver disease (ALD), a condition comprising a spectrum of clinical signs and morphological changes, ranging from fatty liver (steatosis) to more severe forms of chronic liver injury. Secondary cofactors, such as nutritional and hepatotoxic comorbid conditions, can also contribute to liver disease development. Here we investigated the effects in the progression of ALD following short-term exposure to diet high in refined carbohydrates (HC), a high-sugar and -butter (HSB) hypercaloric diet and acute Et-OH consumption. HSB diet increased the body weight (BW) and adiposity independently of acute Et-OH consumption. HC diet did not affect BW but increased the adiposity, while acute Et-OH alone did not affect BW and adiposity. All groups of mice developed steatosis except the control group. Exposure to acute Et-OH and HSB diet increased the number of neutrophils and macrophages, and apoptosis in the liver. This combination also increased the number of circulating neutrophils and reduced mononuclear cells in the blood. Thus, short-term exposure to HSB diet and acute Et-OH intake is linked to increased liver injury. These findings offer important clues to understand the hepatic injuries associated with short exposure to hypercaloric diets and acute Et-OH.

Modulation of Binge-like Ethanol Consumption by IL-10 Signaling in the Basolateral Amygdala.

Excessive ethanol consumption alters the neuroimmune system and particularly impacts the cytokine milieu of the CNS. Cytokine dysregulation has been shown to underlie addictive-like behaviors including alcohol abuse; however, many studies focus primarily on the proinflammatory cytokine profile duringalcohol dependence. The current study furthers this research by determining the impact of excessive ethanol consumption on interleukin-10 (IL-10) and interleukin-4 (IL-4) activity in a model of non-dependent binge consumption called the "drinking in the dark" (DID) paradigm. Furthermore, the ability of IL-10 to modulate ethanol consumption was tested using site-directed pharmacology. Immunohistochemistry analyses determined that ethanol decreased IL-10 by 50% in the basolateral amygdala (BLA) but had no effect on IL-4. Neither IL-10 nor IL-4, however, were altered in the central amygdala (CEA). Enzyme linked immunosorbent assays confirmed that IL-10 was decreased in the amygdala but not in the serum, suggesting changes of this cytokine with the DID paradigm arerestricted to the central nervous system. Finally, bilateral infusions of IL-10 into the BLA, but not CeA, reduced binge-like drinking and corresponding blood ethanol concentrations without impacting either locomotor activity or anxiety-like behavioral correlates. Together, these data support the idea that alcohol abuse dysregulates specific anti-inflammatory cytokines; however, ameliorating alcohol-induced effects on cytokines, like IL-10, may prove to be an effective therapy in curbing excessive consumption.

Binge Drinking Decreases Corticotropin‐Releasing Factor‐Binding Protein Expression in the Medial Prefrontal Cortex of Mice

Dysregulation of the corticotropin-releasing factor (CRF) system has been observed in rodent models of binge drinking, with a large focus on CRF receptor 1 (CRF-R1). The role of CRF-binding protein (CRF-BP), a key regulator of CRF activity, in binge drinking is less well understood. In humans, single-nucleotide polymorphisms in CRHBP are associated with alcohol use disorder and stress-induced alcohol craving, suggesting a role for CRF-BP in vulnerability to alcohol addiction. The role and regulation of CRF-BP in binge drinking were examined in mice exposed to the drinking in the dark (DID) paradigm. Using insitu hybridization, the regulation of CRF-BP, CRF-R1, and CRF mRNA expression was determined in the stress and reward systems of C57BL/6J mice after repeated cycles of DID. To determine the functional role of CRF-BP in binge drinking, CRF-BP knockout (CRF-BP KO) mice were exposed to 6 cycles of DID, during which alcohol consumption was measured and compared to wild-type mice. CRF-BP mRNA expression was significantly decreased in the prelimbic (PL) and infralimbic medial prefrontal cortex (mPFC) of C57BL/6J mice after 3 cycles and in the PL mPFC after 6 cycles of DID. No significant changes in CRF or CRF-R1 mRNA levels were observed in mPFC, ventral tegmental area, bed nucleus of the stria terminalis, or amygdala after 3 cycles of DID. CRF-BP KO mice do not show significant alterations in drinking compared to wild-type mice across 6 cycles of DID. These results reveal that repeated cycles of binge drinking alter CRF-BP mRNA expression in the mPFC, a region responsible for executive function and regulation of emotion and behavior, including responses to stress. We observed a persistent decrease in CRF-BP mRNA expression in the mPFC after 3 and 6 DID cycles, which may allow for increased CRF signaling at CRF-R1 and contribute to excessive binge-like ethanol consumption.

Salvia miltiorrhiza Bunge Blocks Ethanol-Induced Synaptic Dysfunction through Regulation of NMDA Receptor-Dependent Synaptic Transmission.

Consumption of high doses of ethanol can lead to amnesia, which often manifests as a blackout. These blackouts experienced by ethanol consumers may be a major cause of the social problems associated with excess ethanol consumption. However, there is currently no established treatment for preventing these ethanol-induced blackouts. In this study, we tested the ethanol extract of the roots of Salvia miltiorrhiza (SM) for its ability to mitigate ethanol-induced behavioral and synaptic deficits. To test behavioral deficits, an object recognition test was conducted in mouse. In this test, ethanol (1 g/kg, i.p.) impaired object recognition memory, but SM (200 mg/kg) prevented this impairment. To evaluate synaptic deficits, NMDA receptor-mediated excitatory postsynaptic potential (EPSP) and long-term potentiation (LTP) in the mouse hippocampal slices were tested, as they are known to be vulnerable to ethanol and are associated with ethanol-induced amnesia. SM (10 and 100 μg/ml) significantly ameliorated ethanol-induced long-term potentiation and NMDA receptor-mediated EPSP deficits in the hippocampal slices. Therefore, these results suggest that SM prevents ethanol-induced amnesia by protecting the hippocampus from NMDA receptor-mediated synaptic transmission and synaptic plasticity deficits induced by ethanol.