Loss of control over the ethanol consumption: differential transcriptional regulation in prefrontal cortex.

Alcohol use disorder (AUD) is a complex multifactorial disease with heritability of ∼50% and corresponds to the state in which the body triggers a reinforcement or reward compulsive behavior due to ethanol consumption, even when faced with negative consequences. Although several studies have shown the impact of high ethanol intake on the prefrontal cortex (PFC) gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with relapsing. In this study, we used a chronic three-bottle free-choice mouse model to investigate the PFC transcriptome in three different groups of mice drinkers: 'Light drinkers' (preference for water throughout the experiment); 'Heavy drinkers' (preference for ethanol with a non-compulsive intake), and 'Inflexible drinkers' (preference for ethanol with a compulsive drinking component). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the PFC, a brain region critical for the development and maintenance of alcohol addiction. We found that the Camk2a gene showed a downregulated profile only in the Inflexible when compared to the Light drinkers group, the Camk2n1 and Pkp2 genes showed an upregulated profile only in the Inflexible drinkers when compared to the Control group, and the Gja1 gene showed an upregulated profile in the Light and Inflexible drinkers when compared to the Control group. These different transcription patterns have been associated to the presence of alcohol, in the Camk2n1 and Gja1 genes; to the amount of ethanol consumed, in the Camk2a gene; and to the loss of control in the alcohol consumption, in the Pkp2 gene. Here, we provide, for the first time, the potential involvement of the Pkp2 gene in the compulsivity and loss of control over the voluntary ethanol consumption.

Possible involvement of ACSS2 gene in alcoholism.

Alcoholism is a psychiatric disorder that composes one of the principal causes of health disabilities in the world population. Furthermore, the available pharmacotherapy is limited. Therefore, this research was carried out to better understand the basis of the underlying neurobiological processes of this disorder and to discover potential therapeutic targets. Real-time PCR analysis was performed in the amygdala nuclei region of the brain of mice exposed to a chronic three-bottle free-choice model (water, 5 and 10% v/v ethanol). Based on individual ethanol intake, the mice were classified into three groups: "compulsive-like" (i.e., ethanol intake not affected by quinine adulteration), "ethanol-preferring" and "ethanol non-preferring". A fourth group had access only to tap water (control group). The candidate gene ACSS2 was genotyped in human alcoholics by real-time polymerase chain reaction using the markers rs6088638 and rs7266550. Seven genes were picked out (Acss2, Acss3, Acat1, Acsl1, Acaa2, Hadh, and Hadhb) and the mRNA level of the Acss2 gene was increased only in the "compulsive-like" group (p = 0.004). The allele frequency of rs6088638 for the gene ACSS2 was higher in the Alcoholic human group (p = 0.03), although sample size was very small. The gene ACSS2 is associated with alcoholism, suggesting that biochemical pathways where it participates may have a role in the biological mechanisms susceptible to the ethanol effects.

The circling mutant Pcdh15roda is a new mouse model for hearing loss.

Mouse mutagenesis is a key tool for studying gene function and several mutant alleles have been described and constitute mouse models for human hereditary diseases. Genetic hearing loss represents over 50% of all hearing loss cases in children and, due to the heterogeneity of the disorder, there is still a demand for the isolation and characterization of new genes and alleles. Here we report phenotypic and molecular characterization of a new mouse model for hereditary hearing loss. The mutant rodador, isolated by Massironi and colleagues in 2006, presents an autosomal recessive disorder characterized by deafness and balance dysfunction associated with abnormal stereocilia in the inner ear. The mutation was mapped to mouse chromosome 10, and characterization of the gene Pcdh15 revealed an AT-to-GC transition in intron 23 of mutant animals. The alteration led to the switch of a dinucleotide ApA for ApG, creating a novel intronic acceptor splice site, which leads to incorporation of eight intronic bases into the processed mRNA and alteration of the downstream reading frame. In silico analysis indicated that the mutated protein is truncated and lacks two cadherin domains, and the transmembrane and cytoplasmic domains. Real Time PCR analyses revealed a significantly reduced Pcdh15 mRNA level in the brain of mutant mice, which might be due to the mechanism of non-sense mediated decay. In man, mutations in the orthologue PCDH15 cause non-syndromic deafness and Usher Syndrome Type 1F, a genetic disorder characterized by hearing loss and retinitis pigmentosa. Rodador mouse constitutes a new model for studying deafness in these conditions and may help in the comprehension of the pathogeneses of the disease, as well as of the mechanisms involved in the morphogenesis and function of inner ear stereocilia. This is a new ENU-induced allele and the first isolated in a BALB/c background.

Perinatal thiamine restriction affects central GABA and glutamate concentrations and motor behavior of adult rat offspring.

The purposes of the present study were to investigate the effects of perinatal thiamine deficiency, from the 11th day of gestation until the 5th day of lactation, on motor behavior and neurochemical parameters in adult rat offspring, using 3-month-old, adult, male Wistar rats. All rats were submitted to motor tests, using the rotarod and paw print tasks. After behavioral tests, their thalamus, cerebellum and spinal cord were dissected for glutamate and GABA quantifications by high performance liquid chromatography. The thiamine-restricted mothers (RM) group showed a significant reduction of time spent on the rotarod at 25 rpm and an increase in hind-base width. A significant decrease of glutamate concentration in the cerebellum and an increase of GABA concentrations in the thalamus were also observed. For the offspring from control mothers (CM) group there were significant correlations between thalamic GABA concentrations and both rotarod performance and average hind-base width. In addition, for rats from the RM group a significant correlation between stride length and cerebellar GABA concentration was found. These results show that the deficiency of thiamine during an early developmental period affects certain motor behavior parameters and GABA and glutamate levels in specific brain areas. Hence, a thiamine deficiency episode during an early developmental period can induce motor impairments and excitatory and inhibitory neurotransmitter changes that are persistent and detectable in later periods of life.

Reduction of ethanol intake by corticotropin-releasing factor receptor-1 antagonist in "heavy-drinking" mice in a free-choice paradigm.

RATIONALE: We hypothesized that the corticotropin-releasing factor (CRF) system is hyperresponsive in animals with high ethanol intake, which exhibits a reduction of ethanol intake when administered with a CRF1 receptor antagonist. METHODS: Outbred Swiss mice were subjected to a long-term, three-bottle, free-choice paradigm (5 and 10 % [v/v] ethanol and water) that consisted of four phases: acquisition (AC; 10 weeks), withdrawal (W; 2 weeks), reexposure (RE; 2 weeks), and quinine-adulteration (AD; 2 weeks). Based on individual ethanol intake, the mice were classified into three groups: A group, preference for ethanol and persistently high consumption during AD phase; B group, preference for ethanol and a reduction of ethanol intake in the AD phase; and C group; preference for water during all phases. A control group only had access to water. CRF1 receptor messenger RNA (mRNA) levels in the amygdala and the effect of the CRF1 receptor antagonist CP-154,526 on ethanol and water intake in the subgroups were studied. RESULTS: CRF1 transcript levels were higher in the B group than in the control group. The highest dose of CP-154,526 reduced ethanol intake and preference, with no changes in water consumption, in the A group compared with vehicle. The B group exhibited a reduction of both ethanol and water intake, with no changes in preference. The C group exhibited no changes in response to the CRF1 antagonist. CONCLUSIONS: CRF1 receptors appear to be involved in ethanol consumption in mice with high ethanol consumption, and CRF system-mediated neuroadaptations depend on drinking profiles.

A transcriptional study in mice with different ethanol-drinking profiles: possible involvement of the GABA(B) receptor.

Previous studies have suggested that γ-aminobutyric acid-B (GABA(B)) receptor agonists effectively reduce ethanol intake. The quantification using real-time polymerase chain reaction of Gabbr1 and Gabbr2 mRNA from the prefrontal cortex, hypothalamus, hippocampus, and striatum in mice exposed to an animal model of the addiction developed in our laboratory was performed to evaluate the involvement of the GABA(B) receptor in ethanol consumption. We used outbred, Swiss mice exposed to a three-bottle free-choice model (water, 5% v/v ethanol, and 10% v/v ethanol) that consisted of four phases: acquisition (AC), withdrawal (W), reexposure (RE), and quinine-adulteration (AD). Based on individual ethanol intake, the mice were classified into three groups: "addicted" (A group; preference for ethanol and persistent consumption during all phases), "heavy" (H group; preference for ethanol and a reduction in ethanol intake in the AD phase compared to AC phase), and "light" (L group; preference for water during all phases). In the prefrontal cortex in the A group, we found high Gabbr1 and Gabbr2 transcription levels, with significantly higher Gabbr1 transcription levels compared with the C (ethanol-naive control mice), L, and H groups. In the hippocampus in the A group, Gabbr2 mRNA levels were significantly lower compared with the C, L, and H groups. In the striatum, we found a significant increase in Gabbr1 transcription levels compared with the C, L, and H groups. No differences in Gabbr1 or Gabbr2 transcription levels were observed in the hypothalamus among groups. In summary, Gabbr1 and Gabbr2 transcription levels were altered in cerebral areas related to drug taking only in mice behaviorally classified as "addicted" drinkers, suggesting that these genes may contribute to high and persistent ethanol consumption.

Trait anxiety and ethanol: anxiolysis in high-anxiety mice and no relation to intake behavior in an addiction model.

Anxiety has been proposed to play a role in the development of alcohol addiction, but the exact mechanisms by which this occurs remain unclear. The present study aimed to verify the relationship between basal anxiety levels, the anxiolytic-like effect of ethanol, and ethanol intake in mice exposed to an addiction model. In one experiment Swiss mice were characterized as high-anxiety (HA), medium-anxiety (MA), or non-anxiety (NA) in the elevated plus maze and then received saline or ethanol 2 g/kg acutely and chronically and were again exposed to the same test. NA mice decreased while MA mice maintained anxiety indices over the test days, regardless of treatment. HA ethanol-treated mice showed an anxiolytic-like effect, both acutely and chronically, while the saline-treated ones maintained their basal anxiety levels. In another experiment HA and MA mice were exposed to an addiction model based on a 3-bottle free-choice paradigm (ethanol 5% and 10%, and water) consisting of four phases: acquisition (10 weeks), withdrawal (W, 2 weeks), reexposure (2 weeks), and quinine-adulteration (2 weeks). HA and MA control mice had access only to water. Mice were characterized as addicted, heavy-drinker and light-drinker [Fachin-Scheit DJ, Ribeiro AF, Pigatto G, Goeldner FO, Boerngen-Lacerda R. Development of a mouse model of ethanol addiction: naltrexone efficacy in reducing consumption but not craving. J Neural Transm 2006;113:1305-21.]. No difference was observed between HA and MA mice in their preference for and intake of ethanol. No correlation was observed between ethanol intake, during any phase, and anxiety indices measured in the basal tests and during the W phase. The differences in anxiety indices between HA and MA groups persisted in the test performed during ethanol withdrawal, suggesting a "trait" anxiety profile. The data suggest that despite the fact that high anxiety trait levels are important for the anxiolytic-like effects of ethanol, they are not a determining factor for high ethanol intake, at least not under these experimental conditions.