Symposium 1, Sunday Sept. 23rd 2 pm–3.30 pm; Room: Lecture Hall 1
Gene expression and genetic variability in alcohol preferring rat lines: Chairpersons: Hyytia P (Finland), Sommer W (USA)
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Abstract |
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Presentation S1-1
Variation at candidate gene loci and their functional importance in rodent models of ethanol dependence
Bjork K (USA)
Aims. The overall aim of this study is to identify candidate genes in key brain areas for ethanol dependence in genetically selected alcohol-preferring rats and in addition, to search for genetic variation associated with these genes.
Methods. Microarrays and in situ hybridization was used to screen for genes that are differentially expressed in brain areas of genetically selected alcohol-preferring rats (AA and msP lines) compared to their non-preferring counterparts. Expression differences were confirmed using quantitative reverse transcription Real-Time PCR, in situ hybridization or Western blot. To identify cis-regulatory elements driving expression differences screening of the candidate gene promoters for genetic variation was also performed.
Results. Several genes were identified as differentially expressed in alcohol-preferring rats compared to non-preferring rats in the initial microarray screening. Genes with robust differences in several brain regions and involved in biological processes relevant to ethanol dependence were chosen for further analysis. β-arrestin 2 (Arrb2), corticotrophin releasing hormone receptor subtype 1 (Crhr1) and Glutathione S-transferases 
4 (Gsta4) fulfilled both of these criteria. Differential expression of these genes was confirmed using alternative methods. Furthermore, sequence analysis revealed allelic variants of these genes that correlate with altered expression levels.
Conclusions. The combined use of high-throughput methods and rats genetically selected for high ethanol consumption provide a powerful tool to identify candidate genes for ethanol dependence. All of the genes that we could confirm displayed genetic variation at the candidate gene loci which suggests that these genes are mainly cis-regulated. In addition to revealing novel candidate genes for ethanol dependence the approach described above may also provide insight into how these genes are regulated.
Presentation S1-2
Role of brain CRH systems in ethanol dependence
Hansson AC, Rimondini R, Cippitelli A, Terasmaa A, Thorsell A, Ciccocioppo R, Sommer WH, Heilig M, Conrod P, Castellanos N (USA)
Aims. Alcoholism is a chronic, relapsing disorder. It develops through a long history of repeated cycles of intoxication and withdrawal. Over the course of this process, a major shift occurs from positively reinforced, ‘reward drinking’, to negatively reinforced ‘relief drinking’. This in turn has been postulated to result from neuroadaptations that lead to a recruitment of anti-reward systems, i.e. negative reinforcement through stress and fear systems involving extra-hypothalamic corticotrophin-releasing hormone (CRH) systems. We demonstrated the value of this theoretical framework for the design and selection of model phenotypes with good predictive validity for medication development.
Methods. Two animal models of high voluntary ethanol consumption were used: 1) prolonged exposure to repeated cycles of alcohol intoxication and withdrawal in Wistar rats, and 2) the genetically selected Marchigian-Sardinian alcohol-preferring (msP) rat line. Behavioral sensitivity to stress was examined using fear suppression of behavior in the punished drinking (Vogel) conflict test, forced swim stress on voluntary alcohol intake, conditioned emotional response and stress-induced reinstatement of ethanol seeking. The expression of CRH, CRH-R1 and CRH-R2 transcripts within the amygdala complex and hypothalamus was analyzed by in situ hybridization histochemistry. Elevated levels of CRH-R1 transcripts were pharmacologically validated by use of two selective CRH-R1 antagonists (antalarmin and MTIP).
Results. Prolonged exposure to repeated cycles of alcohol intoxication and withdrawal induces a ‘post-dependent’ state, a stable behavioral syndrome characterized by excessive ethanol consumption and behavioral sensitivity to stress. We found that both the elevated self-administration of alcohol and the increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH-R1 subtype of CRH receptors in the amygdala. This converges with findings of a stress-sensitive/anxious behavioral phenotype in the msP rat, accompanied by an innate up-regulation of CRH-R1 receptors in the amygdala and several other brain regions in this line. Using the selective CRH-R1 antagonists antalarmin and MTIP we demonstrate that this accounts for the elevated self-administration of alcohol and the anxious phenotype of both rat models.
Conclusions. Similarly to human alcoholics, the post-dependent state in rats is associated with an enhanced behavioural sensitivity to stress. The increased response to external stressors translates into elevated motivation to consume alcohol in post-dependent animals. High alcohol preference together with increased behavioral sensitivity to stress makes the msP rat a phenocopy of the post-dependent state. A recruitment of intra-amygdala, but not hypothalamic CRH systems seems to be driving both the post-dependent and msP phenotype. In summary, our data support the hypothesis of recruitment of anti-reward systems during the descent into ethanol dependence and their likely importance for maintaining the dependent state. Our findings provide a solid validation of the CRH-R1 receptor as a treatment target.
Presentation S1-3
The use of alcohol preferring rat lines for gene expression profiling in alcohol related disorders
Spanagel R (Germany)
Aims. Our study aimed to identify new candidate genes, which might be involved in (i) alcohol craving and relapse and (ii) in the induction of alcohol-induced organ damage.
Methods. In order to find changes in gene expression following long-term alcohol consumption, we studied gene expression profiles (i) in the striatal dopamine system and (ii) in the pancreas by using DNA microarrays of three different alcohol-preferring rat lines (AA, HAD and P).
Results. Our data revealed an up-regulation of the dopamine D3 receptor (D3R) following one year of voluntary alcohol consumption in the striatum of alcohol preferring rats that was confirmed by qRT-PCR. In the pancreas, among several differentially regulated genes, the regenerating genes Reg1 and Reg3a showed a consistent down-regulation in all 3 lines following long-term alcohol consumption, which was confirmed by qRT-PCR and on the protein level by immunohistochemistry and Western Blot.
Conclusions. We conclude that long-term alcohol consumption leads to an up-regulation of the dopamine D3R that may contribute to alcohol-seeking and relapse. We therefore suggest that selective antagonists of this pharmacological target provide a specific treatment approach to reduce alcohol craving and relapse behavior. Reg genes seem to be critically involved in alcohol-induced pancreatitis and can be used as diagnostic markers as well.
Presentation S1-4
Expression profiling in the prefrontal cortex: Implication for ethanol consumption in animal models of dependence
Sommer WH (USA)
Aims. The AA (Alko, Alcohol) and ANA (Alko, Non-Alcohol) rat lines were among the earliest rodent lines produced by bi-directional selection for ethanol preference. AA rats display high ethanol consumption together with behavioral disinhibition and thus share intermediate phenotypic traits with human early ons et al. coholism. Increased opiodergic tone in the ventral striatum and a deficiency in endocannabinoid signaling in the prefrontal cortex of AA rats may comprise mechanisms leading to the ‘reward seeking’ phenotype of the AA line.
Methods. Because complex behaviors, such as ethanol drinking, are not likely to be controlled by single factors, system-oriented molecular profiling strategies have been employed recently. Microarray based expression analysis of AA and ANA brains and novel data mining strategies provide a system biological view that allows us to formulate a hypothesis on the mechanism underlying selection for ethanol preference.
Results. Two main factors appear active in the selection: a recruitment of signal transduction networks, including MAPK and calcium pathways to mediate ethanol reinforcement and plasticity. The second factor acts on the mitochondrion and most likely provides metabolic flexibility for alternative substrate utilization in the presence of low amounts of ethanol.
Conclusions. These results will be set in context with expression profiling data obtained from other animal models of ethanol preference.