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AbstractThe significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type‐1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol‐induced activation of caspase 3. Together, these data demonstrate that ethanol‐induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis. image Schematic mechanism of action by which ethanol impairs DNA methylation. Studies have demonstrated that ethanol has the capacity to bring epigenetic changes to contribute to the development of fetal alcohol spectrum disorder (FASD). However, the mechanisms are not well studied. P7 ethanol induces the activation of caspase 3 and impairs DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) proteins (→). The inhibition or genetic ablation of cannabinoid receptor type‐1 or inhibition of histone methyltransferase (G9a) by Bix (‐‐‐‐‐) or inhibition of caspase 3 activation by Q‐ quinoline‐Val‐Asp(Ome)‐CH2‐O‐phenoxy (Q‐VD‐OPh) () rescue loss of DNMT1, DNMT3A as well as DNA methylation. Hence, the putative DNMT1/DNMT3A/DNA methylation mechanism may have a potential regulatory role in FASD.

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non‐ionic detergent mixture, Triton CF‐54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM1a, 700; exogenous DS‐fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza‐tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.

Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed inXenopusoocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nm) accumulation into DAT‐expressing oocytes increased significantly in response to ethanol (10 min; 10–100 mm). In two‐electrode voltage‐clamp experiments, DAT‐mediated currents were also enhanced significantly by ethanol (10–100 mm). The magnitude of the ethanol‐induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nm) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT‐mediated substrate translocation and substrate‐associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface.

Abstract— Chronic ethanol ingestion in rats leads to a slow rise in brain alcohol dehydrogenase activity which levels off after 2 weeks at approximately twice the initial activity. The half‐time of the rise is approximately 8 days. Abrupt withdrawal of the ethanol is followed by a rapid decline of the brain alcohol dehydrogenase activity to the normal level with a half‐time of approximately 15 h. The difference in time constants between the rise in enzyme activity during ethanol‐feeding and its decline following withdrawal suggests that the increased enzyme activity is at least in part the result of a reduced rate constant of enzyme degradation in the presence of ethanol. The effect of ethanol on brain alcohol dehydrogenase activity is not altered by supplementation of the diet with carbohydrate or vitamins. The effect is seen only in the cerebral hemispheres and not in the brain‐stem. Acquisition of tolerance to ethanol during chronic ethanol ingestion and its extinction following withdrawal follow almost the same time courses as the changes in brain alcohol dehydrogenase activity.

AbstractChronic ethanol treatment is known to alter the function of the γ‐aminobutyric acidA (GABAA) benzodiazepine receptor complex. To determine if genetic differences in development of ethanol dependence are related to expression of GABAA receptor subunits, we measured whole brain levels of mRNA for the α1α3, α6, γ2s, γ2t, and γ3 receptor subunits in withdrawal seizure‐prone and ‐resistant (WSP and WSR, respectively) mice fed an ethanol‐containing liquid diet or a control diet Brain poly(A)+ RNA was converted to cDNA and amplified by the polymerase chain reaction using primers conserved among GABAA receptor subunits. Quantification was carried out by densitometric analysis of Southern blots generated using subunit‐specific probes. Chronic ethanol treatment decreased the content of α1, mRNA in WSP but not WSR mice and decreased the content of α6 mRNA in WSR but not WSP mice. The content of γ3 mRNA was increased by chronic ethanol in both lines. In untreated mice, the WSP line had lower levels of α3 and α6 mRNA than the WSR line. Thus, a decrease in the content of α1 mRNA is most clearly linked with development of withdrawal signs, although the amounts of α6 and α3 may also be important in the genetic differences between WSP and WSR mice. In contrast, levels of mRNA for γ2S and γ2L subunits do not appear to be altered in ethanol dependence.

AbstractBinge alcohol drinking, a risky pattern of alcohol consumption, has severe consequences toward health and well‐being of an individual, his family, and society. Although, binge drinking has detrimental effects on sleep, underlying mechanisms are unknown. We used adult male C57BL/6J mice and exposed them to a single, 4‐h session of binge alcohol self‐administration, in stress‐free environment, to examine neuronal mechanisms affecting sleep. We first verified binge pattern of alcohol consumption. When allowed to self‐administer alcohol in a non‐stressful environment, mice consumed alcohol in a binge pattern. Next, effect of binge drinking on sleep–wakefulness was monitored. While sleep–wakefulness remained unchanged during drinking session, significant increase in non‐rapid eye movement (NREM) sleep was observed during 4 h of active period post‐binge, followed by increased wakefulness, reduced sleep during subsequent sleep (light) period; although the timing of sleep onset (at lights‐on) remained unaffected. Next, electrophysiological and biochemical indicators of sleep homeostasis were examined using sleep deprivation‐recovery sleep paradigm. Mice exposed to binge drinking did not show an increase in cortical theta power and basal forebrain adenosine levels during sleep deprivation; NREM sleep and NREM delta power did not increase during recovery sleep suggesting that mice exposed to binge alcohol do not develop sleep pressure. Our final experiment examined expression of genes regulating sleep homeostasis following binge drinking. While binge drinking did not affect adenosine kinase and A1 receptor, expression of equilibrative nucleoside transporter 1 (ENT1) was significantly reduced. These results suggest that binge alcohol consumption‐induced down‐regulation of ENT1 expression may disrupt sleep homeostasis and cause sleep disturbances.Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ image

AbstractNicotine and alcohol co‐abuse is highly prevalent, although the underlying causes are unclear. It has been suggested that nicotine enhances pleasurable effects of alcohol while reducing aversive effects. Recently, we reported that nicotine acts via the basal forebrain (BF) to activate nucleus accumbens and increase alcohol consumption. Does nicotine suppress alcohol‐induced aversive effects via the BF? We hypothesized that nicotine may act via the BF to suppress sleep‐promoting effects of alcohol. To test this hypothesis, adult male Sprague–Dawley rats were implanted with sleep‐recording electrodes and bilateral guides targeted toward the BF. Nicotine (75 pmol/500 nL/side) or artificial cerebrospinal fluid (ACSF; 500 nL/side) was microinjected into the BF followed by intragastric alcohol (ACSF + EtOH and NiC + EtOH groups; 3 g/kg) or water (NiC + W and ACSF + W groups; 10 mL/kg) administration. On completion, rats were killed and processed to localize injection sites in the BF. The statistical analysis revealed a significant effect of treatment on sleep‐wakefulness. While rats exposed to alcohol (ACSF + EtOH) displayed strong sleep promotion, nicotine pre‐treatment in the BF (NiC + EtOH) attenuated alcohol‐induced sleep and normalized sleep‐wakefulness. These results suggest that nicotine acts via the BF to suppress the aversive, sleep‐promoting effects of alcohol, further supporting the role of BF in alcohol‐nicotine co‐use.image Alcohol and nicotine are highly co‐abused. The underlying mechanism is unclear. One reason why people use nicotine, a stimulant, with alcohol is to enhance recreational/pleasurable sensations while suppressing alcohol's aversive effects such as sleepiness. We have previously observed that nicotine acts via wake‐promoting basal forebrain to increase alcohol consumption and activate nucleus accumbens, the pleasure center. Here, we demonstrate that nicotine acts via the basal forebrain to suppress sleep‐promoting effects of alcohol further implicating the basal forebrain as a key substrate in nicotine alcohol co‐use.

AbstractAlcohol engages signaling pathways in the brain. Midkine (MDK) is a neurotrophic factor that is over‐expressed in the prefrontal cortex of alcoholics.MDKand one of its receptors, anaplastic lymphoma kinase (ALK), also regulate behavioral responses to ethanol in mice. The goal of this study was to determine whetherMDKandALKexpression and signaling are activated by ethanol. We found that ethanol treatment of neuroblastoma cells increasedMDKandALKexpression. We also assessed activation ofALKby ethanol in cells and found thatALKandALK‐dependent extracellular signal‐regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) phosphorylation increased rapidly with ethanol exposure. Similarly, treatment of cells with recombinantMDKprotein increasedALK,ERKand STAT3 phosphorylation, suggesting that ethanol may utilizeMDKto activateALKsignaling. In support of this, transfection of cells withMDKsiRNAs attenuatedALKsignaling in response to ethanol. Ethanol also activatesERKsignaling in the brain. We found that inhibition ofALKor knockout ofMDKattenuated ethanol‐inducedERKphosphorylation in mouse amygdala. These results demonstrate that ethanol engagesMDKandALKsignaling, which has important consequences for alcohol‐induced neurotoxicity and the regulation of behaviors related to alcohol abuse.imageWe propose that ethanol (a) increases transcription of the anaplastic lymphoma kinase (ALK) and midkine (MDK) genes and (b) rapidly activates extracellular signal‐regulated kinase (pERK1/2) and signal transducer and activator of transcription 3 (pSTAT3) through MDK and ALK. Activation of ALK and MDK signaling by ethanol may alter behavioral responses to ethanol with implications for the development of alcohol use disorders.