Microglial cells are the primary immune effector cells in the brain and play a pivotal role in the neuroinflammatory processes associated with a variety of neurological and pathological disorders. Alcohol consumption induces brain damage, although the neuropathological processes are poorly understood. We previously suggested that ethanol promotes inflammatory processes in the brain, up-regulating inflammatory mediators and signaling pathways associated with IL-1RI/TLR4 receptors. In the present study we investigate whether ethanol induces microglia activation by stimulating TLR4 response and whether this response causes neuronal death and contributes to ethanol-induced neuroinflammatory damage. We demonstrate that ethanol activates microglía and stimulates NF-κB, MAPKs, and MyD88-independent (IFN regulatory factor-3, IFN-β) pathways to trigger the production of inflammatory mediators, causing neuronal death. The inflammatory response induced by ethanol is completely abrogated in microglia of TLR4-deficient mice (TLR4^−/−), thus supporting the role of these receptors in microglia activation and neuronal death. In accord with the in vitro findings, acute ethanol administration induces microglia activation (CD11b⁺ cells) in cerebral cortex of TLR4^+/+ mice, but not in TLR4^−/− mice. Taken together, our results not only provide the first evidence of the critical role of the TLR4 response in the ethanol-induced microglia activation, but also new insight into the basic mechanisms participating in ethanol-induced neuroinflammatory damage.

Adolescence is a developmental period which the risk of drug and alcohol abuse increases. Since mesolimbic dopaminergic system undergoes developmental changes during adolescence, and this system is involved in rewarding effects of drugs of abuse, we addressed the hypothesis that ethanol exposure during juvenile/adolescent period over‐activates mesolimbic dopaminergic system inducing adaptations which can trigger long‐term enduring behavioural effects of alcohol abuse. We treated juvenile/adolescent or adult rats with ethanol (3 g/kg) for two‐consecutive days at 48‐h intervals over 14‐day period. Here we show that intermittent ethanol treatment during the juvenile/adolescence period alters subsequent ethanol intake.In vivomicrodialysis demonstrates that ethanol elicits a similar prolonged dopamine response in the nucleus accumbens of both adolescent and adult animals pre‐treated with multiple doses of ethanol, although the basal dopamine levels were higher in ethanol‐treated adolescents than in adult‐treated animals. Repeated ethanol administration also down‐regulates the expression of DRD2 and NMDAR2B phosphorylation in prefrontal cortex of adolescent animals, but not of adult rats. Finally, ethanol treatment during adolescence changes the acetylation of histones H3 and H4 in frontal cortex, nucleus accumbens and striatum, suggesting chromatin remodelling changes. In summary, our findings demonstrate the sensitivity of adolescent brain to ethanol effects on dopaminergic and glutamatergic neurotransmission, and suggest that abnormal plasticity in reward‐related processes and epigenetic mechanisms could contribute to the vulnerability of adolescents to alcohol addiction.

Activated astroglial cells are implicated in neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators and cytokines have been proposed to play a key role in glial cell-related brain damage. Cytokine production seems to be initiated by signaling through TLR4/type I IL-1R (IL-1RI) in response to their ligands, LPS and IL-1β, playing vital roles in innate host defense against infections, inflammation, injury, and stress. We have shown that glial cells are stimulated by ethanol, up-regulating cytokines and inflammatory mediators associated with TLR4 and IL-1RI signaling pathways in brain, suggesting that ethanol may contribute to brain damage via inflammation. We explore the possibility that ethanol, in the absence of LPS or IL-1β, triggers signaling pathways and inflammatory mediators through TLR4 and/or IL-1RI activation in astrocytes. We show in this study that ethanol, at physiologically relevant concentrations, is capable of inducing rapid phosphorylation within 10 min of IL-1R-associated kinase, ERK1/2, stress-activated protein kinase/JNK, and p38 MAPK in astrocytes. Then an activation of NF-κB and AP-1 occurs after 30 min of ethanol treatment along with an up-regulation of inducible NO synthase and cyclooxygenase-2 expression. Finally, we note an increase in cell death after 3 h of treatment. Furthermore, by using either anti-TLR4- or anti-IL-1RI-neutralizing Abs, before and during ethanol treatment, we inhibit ethanol-induced signaling events, including NF-κB and AP-1 activation, inducible NO synthase, and cyclooxygenase-2 up-regulation and astrocyte death. In summary, these findings indicate that both TLR4 and IL-1RI activation occur upon ethanol treatment, and suggest that signaling through these receptors mediates ethanol-induced inflammatory events in astrocytes and brain.