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Adolescents frequently engage in risky behaviours such as binge drinking. Binge drinking, in turn, perturbs neurodevelopment reinforcing reward seeking behaviour in adulthood. Current animal models are limited in their portrayal of this behaviour and the assessment of neuroimmune involvement (specifically the role of Toll-like receptor 4 (TLR4)). Therefore, the aims of this project were to develop a more relevant animal model of adolescent alcohol exposure and to characterise its effects on TLR4 signalling and alcohol-related behaviours later life. Balb/c mice received a short (P22-P25), low dose alcohol binge during in early adolescence, and underwent tests to investigate anxiety (elevated plus maze), alcohol seeking (conditioned place preference) and binge drinking behaviour (drinking in the dark) in adulthood. Four doses of alcohol during adolescence increased alcohol-induced conditioned place preference and alcohol intake in adulthood. However, this model did not affect basal elevated plus maze performance. Subsequent analysis of nucleus accumbal mRNA, revealed increased expression of TLR4-related mRNAs in mice who received alcohol during adolescence. To further elucidate the role of TLR4, (+)-Naltrexone, a biased TLR4 antagonist was administered 30 min before or after the adolescent binge paradigm. When tested in adulthood, (+)-Naltrexone treated mice exhibited reduced alcohol intake however, alcohol seeking and anxiety behaviour was unaltered. This study highlights that even a small amount of alcohol, when given during a critical neurodevelopmental period, can potentiate alcohol-related behaviours and TLR4 activation later in life. Interestingly, attenuation of TLR4 before or after adolescent alcohol exposure reduced only binge alcohol intake in adulthood.

The ability of drugs of abuse to increase mesolimbic levels of dopamine is a characteristic associated with their rewarding effects. Exactly how these effects are produced by different substances is not as well characterised. Our previous work in rats has demonstrated that accumbal glycine receptors (GlyRs) are involved in mediating the dopamine-activating effects of ethanol, and in modulating ethanol intake. In this study the investigation of GlyR involvement was extended to include several different drugs of abuse. By using microdialysis and electrophysiology we compared effects of addictive drugs, with and without the GlyR antagonist strychnine, on dopamine levels and neurotransmission in nucleus accumbens. The dopamine-increasing effect of systemic ethanol and the drug-induced change in neurotransmission in vitro, as measured by microdialysis and field potential recordings, were dependent on GlyRs in nAc. Accumbal GlyRs were also involved in the actions of tetrahydrocannabinol and nicotine, but not in those of cocaine or morphine. These data indicate that accumbal GlyRs play a key role in ethanol-induced dopamine activation and contribute also to that of cannabinoids and nicotine.

Genetic influences on the predisposition to complex behavioral or physiological traits can reflect genetic polymorphisms that lead to altered gene product function, and/or variations in gene expression levels. We have explored quantitative variations in an animal's alcohol consumption, using a genetical genomic/phenomic approach. In our studies, gene expression is correlated with amount of alcohol consumed, and genomic regions that regulate the alcohol consumption behavior and the quantitative levels of gene expression (behavioral and expression quantitative trait loci [QTL]) are determined and used as a filter to identify candidate genes predisposing the behavior. We determined QTLs for alcohol consumption using the LXS panel of recombinant inbred mice. We then identified genes that were: 1) differentially expressed between five high and five low alcohol-consuming lines or strains of mice; and 2) were physically located in, or had an expression QTL (eQTL) within the alcohol consumption QTLs. Comparison of mRNA and protein levels in brains of high and low alcohol consuming mice led us to a bioinformatic examination of potential regulation by microRNAs of an identified candidate transcript, Gnb1 (G protein beta subunit 1). We combined our current analysis with our earlier work identifying candidate genes for the alcohol consumption trait in mice, rats and humans. Our overall analysis leads us to postulate that the activity of the GABAergic system, and in particular GABA release and GABA receptor trafficking and signaling, which involves G protein function, contributes significantly to genetic variation in the predisposition to varying levels of alcohol consumption. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Recent electrophysiological evidence suggests that ethanol simultaneously exerts opposite effects on the activity of dopamine (DA) neurons in the ventral tegmental area (VTA) through two parallel mechanisms, one promoting and the other reducing the GABA release onto VTA DA neurons. Here we explore the possible behavioural implications of these findings by investigating the role displayed by acetaldehyde (the main metabolite of ethanol) and the non-metabolized fraction of ethanol in motor activity of rats. We analyse the appearance of motor activation or depression after intra-VTA administration of ethanol in rats subjected to different pharmacological pre-treatments designed to preferentially test either the effects of acetaldehyde or the non-metabolized ethanol. Motor activity was evaluated after intra-VTA administration of 35 nmol of ethanol, an apparently ineffective dose that does not modify the motor activity of animals. Pharmacological pre-treatments were used in order to either increase (cyanamide, 10 mg/kg, ip) or decrease (D-penicillamine, 50 mg/kg, ip and sodium azide, 7 mg/kg, ip) acetaldehyde levels in the VTA. Pre-treatments aimed to augment acetaldehyde, increased motor activity of rats. Otherwise, pre-treatments intended to decrease local acetaldehyde levels evoked significant reductions in motor activity that were prevented by the local blockade (bicuculline, 17.5 pmol) of the GABAA receptors. Our findings suggest that the brain-generated acetaldehyde is involved in the stimulant effects of ethanol, whereas the non-biotransformed fraction of ethanol, acting through the GABAA receptors, would account for the depressant effects. The present behavioural findings suggest that ethanol dually modulates the activity of DA neurons.

Dysfunction of N-methyl-d-aspartate receptor (NMDAR) signaling in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng), a calmodulin-binding protein, is exclusively expressed in the post-synapse, and mediates NMDAR driven synaptic plasticity by regulating the calcium-calmodulin (Ca2+-CaM) pathway. To study the functional role of Ng in AUD, we administrated behavior tests including Pavlovian instrument transfer (PIT), operant conditioning, and rotarod test using Ng null mice (Ng-/- mice). We used adeno-associated virus (AAV)-mediated Ng expression and pharmacological manipulation to validate behavioral responses in Ng-/- mice. The results from our multidisciplinary approaches demonstrated that deficit of Ng increases tolerance to NMDAR inhibition and elicit faster cue reactivity during PIT without changes in ethanol reward. Operant conditioning results demonstrated that Ng-/- mice self-administered significantly more ethanol and displayed reduced sensitivity to aversive motivation. We identified that ethanol exposure decreases mGluR5 (metabotropic glutamate receptor 5) expression in the NAc of Ng-/- mice and pharmacological inhibition of mGluR5 reverses NMDAR desensitization in Ng-/- mice. Together these findings specifically suggest that accumbal Ng plays an essential role in the counterbalance between NMDAR and mGluR5 signaling; which alters NMDAR resistance, and thereby altering aversive motivation for ethanol and may ultimately contribute to susceptibility for alcohol addiction.

Abstract Treatment of rats with ethanol for 3 weeks resulted in a significant increase in δ-opiate receptor binding whereas no change in μ-opiate receptor binding was observed. Scatchard analysis showed an increase in δ-receptor affinity without a change in the receptor density. Acute treatment with ethanol did not alter receptor characteristics. The data provide evidence that δ- and μ-opiate receptor sites can be differentially modulated in vivo.

Prenatal ethanol exposure can damage the developing nervous system, producing long-lasting impairments in both brain structure and function. In this study we analyzed how exposure to this teratogen during the period of brain development affects the intracellular redox state in the brain as well as the development of anxiety- and depressive-like phenotypes. Furthermore, we also tested whether aerobic exercise might have therapeutic potential for fetal alcohol spectrum disorders (FASD) by increasing neuronal antioxidant capacity and/or by alleviating ethanol-induced behavioral deficits. Sprague-Dawley rats were administered ethanol across all three-trimester equivalents (i.e., throughout gestation and during the first 10 days of postnatal life). Ethanol-exposed and control animals were assigned to either sedentary or running groups at postnatal day (PND) 48. Runners had free access to a running wheel for 12 days and at PND 60 anxiety- and depressive-like behaviors were assessed. Perinatal ethanol exposure resulted in the occurrence of depressive and anxiety-like behaviors in adult rats without affecting their locomotor activity. Voluntary wheel running reversed the depressive-like behaviors in ethanol-exposed males, but not in ethanol-exposed females. Levels of lipid peroxidation and protein oxidation were significantly increased in the hippocampus and cerebellum of ethanol-exposed rats, and there was a concomitant reduction in the levels of the endogenous antioxidant glutathione. Voluntary exercise was able to reverse the deficits in glutathione both in ethanol-exposed males and females. Thus, while voluntary physical exercise increased glutathione levels in both sexes, its effects at the behavioral level were sex dependent, with only ethanol-exposed male runners showing a decrease in depressive-like behaviors.

The antioxidant enzyme catalase by reacting with H(2)O(2), forms the compound known as compound I (catalase-H(2)O(2)). This compound is able to oxidise ethanol to acetaldehyde in the CNS. It has been demonstrated that 3-nitropropionic acid (3-NPA) induces the activity of the brain catalase-H(2)O(2) system. In this study, we tested the effect of 3-NPA on both the brain catalase-H(2)O(2) system and on the acute locomotor effect of ethanol. To find the optimal interval for the 3-NPA-ethanol interaction mice were treated with 3-NPA 0, 45, 90 and 135min before an ethanol injection (2.4mg/kg). In a second study, 3-NPA (0, 15, 30 or 45mg/kg) was administered SC to animals 90min before saline or several doses of ethanol (1.6 or 2.4g/kg), and the open-field behaviour was registered. The specificity of the effect of 3-NPA (45mg/kg) was evaluated on caffeine (10mg/kg IP) and cocaine (4mg/kg)-induced locomotion. The prevention of 3-NPA effects on both ethanol-induced locomotion and brain catalase activity by L-carnitine, a potent antioxidant, was also studied. Nitropropionic acid boosted ethanol-induced locomotion and brain catalase activity after 90min. The effect of 3-NPA was prevented by l-carnitine administration. These results indicate that 3-NPA enhanced ethanol-induced locomotion by increasing the activity of the brain catalase system.