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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.

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.

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'.

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'.

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.

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.

Acute ethanol exposure is known to stimulate the dopamine system; however, chronic exposure has been shown to downregulate the dopamine system. In rodents, chronic intermittent exposure (CIE) to ethanol also increases negative affect during withdrawal, such as, increases in anxiety- and depressive-like behavior. Moreover, CIE exposure results in increased ethanol drinking and preference during withdrawal. Previous literature documents reductions in CIE-induced anxiety-, depressive-like behaviors and ethanol intake in response to kappa opioid receptor (KOR) blockade. KORs are located on presynaptic dopamine terminals in the nucleus accumbens (NAc) and inhibit release, an effect which has been linked to negative affective behaviors. Previous reports show an upregulation in KOR function following extended CIE exposure; however it is not clear whether there is a direct link between KOR upregulation and dopamine downregulation during withdrawal from CIE. This study aimed to examine the effects of KOR modulation on dopamine responses to ethanol of behaving mice exposed to air or ethanol vapor in a repeated intermittent pattern. First, we showed that KORs have a greater response to an agonist after moderate CIE compared to air exposed mice using ex vivo fast scan cyclic voltammetry. Second, using in vivo microdialysis, we showed that, in contrast to the expected increase in extracellular levels of dopamine following an acute ethanol challenge in air exposed mice, CIE exposed mice exhibited a robust decrease in dopamine levels. Third, we showed that blockade of KORs reversed the aberrant inhibitory dopamine response to ethanol in CIE exposed mice while not affecting the air exposed mice demonstrating that inhibition of KORs "rescued" dopamine responses in CIE exposed mice. Taken together, these findings indicate that augmentation of dynorphin/KOR system activity drives the reduction in stimulated (electrical and ethanol) dopamine release in the NAc. Thus, blockade of KORs is a promising avenue for developing pharmacotherapies for alcoholism.

Acute ethanol exposure is known to stimulate the dopamine system; however, chronic exposure has been shown to downregulate the dopamine system. In rodents, chronic intermittent exposure (CIE) to ethanol also increases negative affect during withdrawal, such as, increases in anxiety- and depressive-like behavior. Moreover, CIE exposure results in increased ethanol drinking and preference during withdrawal. Previous literature documents reductions in CIE-induced anxiety-, depressive-like behaviors and ethanol intake in response to kappa opioid receptor (KOR) blockade. KORs are located on presynaptic dopamine terminals in the nucleus accumbens (NAc) and inhibit release, an effect which has been linked to negative affective behaviors. Previous reports show an upregulation in KOR function following extended CIE exposure; however it is not clear whether there is a direct link between KOR upregulation and dopamine downregulation during withdrawal from CIE. This study aimed to examine the effects of KOR modulation on dopamine responses to ethanol of behaving mice exposed to air or ethanol vapor in a repeated intermittent pattern. First, we showed that KORs have a greater response to an agonist after moderate CIE compared to air exposed mice using ex vivo fast scan cyclic voltammetry. Second, using in vivo microdialysis, we showed that, in contrast to the expected increase in extracellular levels of dopamine following an acute ethanol challenge in air exposed mice, CIE exposed mice exhibited a robust decrease in dopamine levels. Third, we showed that blockade of KORs reversed the aberrant inhibitory dopamine response to ethanol in CIE exposed mice while not affecting the air exposed mice demonstrating that inhibition of KORs "rescued" dopamine responses in CIE exposed mice. Taken together, these findings indicate that augmentation of dynorphin/KOR system activity drives the reduction in stimulated (electrical and ethanol) dopamine release in the NAc. Thus, blockade of KORs is a promising avenue for developing pharmacotherapies for alcoholism.