María Pascual

Repeated alcohol administration during adolescence causes changes in the mesolimbic dopaminergic and glutamatergic systems and promotes alcohol intake in the adult rat.

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 vivo microdialysis 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.

Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats

Adolescent brain development seems to be important for the maturation of brain structures and behaviour. Intermittent binge ethanol drinking is common among adolescents, and this type of drinking can induce brain damage. Because we have demonstrated that chronic ethanol treatment induces inflammatory processes in the brain, we investigate whether intermittent ethanol intoxication enhances cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) in adolescent rats, and whether these mediators induce brain damage and cause permanent cognitive dysfunctions. Adolescent rats were exposed to ethanol (3.0 g/kg) for two consecutive days at 48‐h intervals over 14 days. Levels of COX‐2, iNOS and cell death were assessed in the neocortex, hippocampus and cerebellum 24 h after the final ethanol administration. The following day or 20 days after the final injection (adult stage), animals were tested for different behavioural tests (conditional discrimination learning, rotarod, object recognition, beam‐walking performance) to assess cognitive and motor functions. Our results show that intermittent ethanol intoxication upregulates COX‐2 and iNOS levels, and increases cell death in the neocortex, hippocampus and cerebellum. Furthermore, animals treated with ethanol during adolescence exhibited behavioural deficits that were evident at the end of ethanol treatments and at the adult stage. Administration of indomethacin, a COX‐2 inhibitor, abolishes the induction of COX‐2 and iNOS expression and cell death, preventing ethanol‐induced behavioural deficits. These findings indicate that binge pattern exposure to ethanol during adolescence induces brain damage by inflammatory processes and causes long‐lasting neurobehavioural consequences. Accordingly, administering indomethacin protects against ethanol‐induced brain damage and prevents detrimental ethanol effects on cognitive and motor processes.

Critical role of TLR4 response in the activation of microglia induced by ethanol.

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.

Chronic ethanol treatment enhances inflammatory mediators and cell death in the brain and in astrocytes.

Inflammatory processes and cytokine expression have been implicated in the pathogenesis of several neurodegenerative disorders. Chronic ethanol intake induces brain damage, although the mechanisms involved in this effect are not well understood.

Involvement of TLR4/Type I IL-1 Receptor Signaling in the Induction of Inflammatory Mediators and Cell Death Induced by Ethanol in Cultured Astrocytes

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.

Glia and fetal alcohol syndrome

Glial cells and their interactions with neurons play vital roles during the ontogeny of the nervous system and in the adult brain. Alcohol intake during pregnancy can cause mental retardation and neurobehavioral disorders as well as fetal alcohol syndrome (FAS). Clinical and experimental evidence indicate that in utero alcohol exposure induces structural and functional abnormalities in gliogenesis and in glial-neuronal interactions, suggesting a potential role of glial cells on ethanol-induced developmental brain abnormalities. In vivo studies have shown ethanol-associated alterations in the migration of neurons and radial glial as well as in astrogliogenesis and myelin development. In astrocytes in primary culture, ethanol has been found to (1) impair cell growth and differentiation, (2) decrease the levels of glialfibrillary acidic protein or GFAP (an astrocyte marker) and its gene expression and (3) interfere with the stimulatory effect of trophic factors affecting their release and receptor expression. Evidence also suggests that ethanol affects intracellular protein trafficking, which may mediate some effects of ethanol on astroglial cells. These findings suggest that glial cells are target of ethanol toxicity during brain development and may underlie the neurodevelopmental abnormalities observed after in utero alcohol exposure and in FAS.

Ethanol exposure enhances cell death in the developing cerebral cortex: role of brain-derived neurotrophic factor and its signaling pathways.

Exposure to ethanol during fetal development induces brain damage, causing cell loss in several brain areas and affecting synaptic connections. Because neurotrophin signaling plays an important role in neuronal survival and differentiation, we have investigated the effect of ethanol exposure on cell death in the developing cerebral cortex and whether this effect correlates with alterations in brain‐derived neurotrophic factor (BDNF) levels, expression of its receptors, TrkB, and its signaling. We report that chronic ethanol intake during gestation and lactation enhances natural cell death and induces cell necrosis, decreases BDNF levels, and increases the ratio of the truncated to full‐length TrkB mRNA receptors during postnatal developing cerebral cortex. Furthermore, we provide evidence that during brain development BDNF activates the extracellular signal‐regulated kinases (ERK1 and ERK2) and the phosphoinoside‐3‐kinase (PI‐3‐K/Akt) pathways. However, BDNF‐induced cell signaling throughout the above‐mentioned survival pathways is significantly reduced by ethanol exposure. These findings suggest that ethanol‐induced alterations in BDNF availability and in its receptor function might impair intracellular signaling pathways involved in cell survival, growth, and differentiation, leading to enhanced natural cell death during cerebral cortex development.

Impact of TLR4 on behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage

Toll-like receptors (TLRs) play an important role in the innate immune response, and emerging evidence indicates their role in brain injury and neurodegeneration. Our recent results have demonstrated that ethanol is capable of activating glial TLR4 receptors and that the elimination of these receptors in mice protects against ethanol-induced glial activation, induction of inflammatory mediators and apoptosis. This study was designed to assess whether ethanol-induced inflammatory damage causes behavioral and cognitive consequences, and if behavioral alterations are dependent of TLR4 functions. Here we show in mice drinking alcohol for 5months, followed by a 15-day withdrawal period, that activation of the astroglial and microglial cells in frontal cortex and striatum is maintained and that these events are associated with cognitive and anxiety-related behavioral impairments in wild-type (WT) mice, as demonstrated by testing the animals with object memory recognition, conditioned taste aversion and dark and light box anxiety tasks. Mice lacking TLR4 receptors are protected against ethanol-induced inflammatory damage, and behavioral associated effects. We further assess the possibility of the epigenetic modifications participating in short- or long-term behavioral effects associated with neuroinflammatory damage. We show that chronic alcohol treatment decreases H4 histone acetylation and histone acetyltransferases activity in frontal cortex, striatum and hippocampus of WT mice. Alterations in chromatin structure were not observed in TLR4(-/-) mice. These results provide the first evidence of the role that TLR4 functions play in the behavioral consequences of alcohol-induced inflammatory damage and suggest that the epigenetic modifications mediated by TLR4 could contribute to short- or long-term alcohol-induced behavioral or cognitive dysfunctions.

Changes in histone acetylation in the prefrontal cortex of ethanol-exposed adolescent rats are associated with ethanol-induced place conditioning.

Alcohol drinking during adolescence can induce long-lasting effects on the motivation to consume alcohol. Abnormal plasticity in reward-related processes might contribute to the vulnerability of adolescents to drug addiction. We have shown that binge-like ethanol treatment in adolescent rats induces alterations in the dopaminergic system and causes histone modifications in brain reward regions. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced alterations in gene expression and behavior, we addressed the hypothesis that ethanol is capable of inducing transcriptional changes by histone modifications in specific gene promoters in adolescent brain reward regions, and whether these events are associated with acquisition of place conditioning. After treating juvenile and adult rats with intermittent ethanol administration, we found that ethanol treatment upregulates histone acetyl transferase (HAT) activity in adolescent prefrontal cortex and increases histone (H3 or H4) acetylation and H3(K4) dimethylation in the promoter region of cFos, Cdk5 and FosB. Inhibition of histone deacetylase by sodium butyrate before ethanol injection enhances both up-regulation of HAT activity and histone acetylation of cFos, Cdk5 and FosB. Furthermore, co-administration of sodium butyrate with ethanol prolongs the extinction of conditioned place aversion and increased the reinstatement effects of ethanol in ethanol-treated adolescents, but not in ethanol-treated adult rats. These results indicate that ethanol exposure during adolescence induces chromatin remodeling, changes histone acetylation and methylation, and modify the effects of ethanol on place conditioning. They also suggest that epigenetic mechanisms might open up avenues to new treatments for binge drinking-induced drug addiction during adolescence.

Ethanol-induced iNOS and COX-2 expression in cultured astrocytes via NF-κB

: The CNS is particularly susceptible to the effects of alcohol and toxicity. Astrocytes are immunoactive cells, and the activation of these cells is associated with several neurodegenerative disorders. By using cultured cortical astrocytes, we show that a short ethanol treatment (100 mM) is able to up-regulate both cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, and that these effects are regulated via nuclear factor kappa B (NF-kappa B) as revealed by the inhibition of NF-kappa B activation with pyrrolidine dithiocarbamate (PDTC) or BAY 11-7082. These results suggest that ethanol is able to induce inflammatory mediators in astrocytes through the NF-kappa B activation.