Sardinian Alcohol-nonpreferring Rats Research Articles

Five mutations in the GABA A α6 gene 5′ flanking region are associated with a reduced basal and ethanol-induced α6 upregulation in mutated Sardinian alcohol non-preferring rats

The presence of four nucleotide changes and a three base-pair deletion in the GABA A α6-subunit promoter is described in Sardinian alcohol non-preferring rats, selectively bred for their ethanol aversion. These mutations are associated with the R100Q α6 intragenic mutation that was previously characterized in the same animals. The possibility that these mutated nucleotides alter the ethanol-induced upregulation of the α6 gene was investigated by measuring cerebellar α6 mRNA levels after a chronic ethanol liquid diet in sNP rat. Real-time quantitative PCR showed an increased α6 gene expression after ethanol ingestion in normal and mutated rats. However, lower amounts of α6 mRNA levels were detected both in control and in ethanol-treated sNP rats carrying the five promoter and the intragenic mutations in a homozygous state. Using the electromobility shift assay, specific DNA binding sites were found in cerebellar extracts of the α6 regions comprising the five mutations. These results suggest that one or more of the mutated binding sites that were found in the 5′ flanking α6 region may be a consensus sequence for regulatory factors which are responsible for both basal and ethanol-induced α6 gene expression.

Characterization of wild-type (R100R) and mutated (Q100Q) GABA A α 6 subunit in Sardinian alcohol non-preferring rats (sNP)

Sardinian alcohol non-preferring (sNP) rats, selected for their low ethanol preference and consumption, carry a point mutation (R100Q) in the gene coding for GABA A receptor α 6 subunit, which becomes more sensitive to diazepam-evoked GABA currents. We performed binding studies in the cerebellum of normal (RR) and mutated (QQ) sNP rats using [ 3H]Ro 15-4513, an inverse agonist for the benzodiazepine site which binds both diazepam insensitive and diazepam sensitive sites. Saturation curves performed on cerebellar membrane from genotyped rats indicated an higher affinity of [ 3H]Ro 15-4513 for GABA A receptors in QQ with respect to RR rats ( K d values 4.0±0.67 and 6.24±0.95 nM, respectively), with similar B max values (3.5±0.25 and 3.9±0.39 pmol/mg protein, respectively). Diazepam displacement curves showed a two component model for both genotypes, with similar K i1 values for QQ and RR (3.6±0.62 and 4.9±0.33 nM, respectively). In QQ rats diazepam is able to completely displace [ 3H]Ro 15-4513 ( K i2=1.48±0.27 μM), while in RR rats the diazepam sensitive sites are still present ( K i2>10 μM). The basal mRNA and protein expression level of the α 6 subunit were similar in RR and QQ rats. The electrophysiological profile of oocytes of Xenopus laevis injected with cerebellar synaptosomes showed that ethanol positively modulated GABA-evoked currents significantly more in QQ than in RR rats. These data contribute to the characterization of the function of GABA A α 6 subunit and its involvement in determining alcohol related behavior.

Differences in ethanol-induced conditioned taste aversion in Sardinian alcohol-preferring and Sardinian alcohol-nonpreferring rats

In the present study, we investigated whether aversion to the pharmacological effects of ethanol developed to a differential extent in selectively bred Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats, and whether this different response was consistent with their genetically determined differences in ethanol preference and consumption. To this purpose, a conditioned taste aversion paradigm was used. Male sP and sNP rats were exposed to five sessions in which a 20-min availability of a saccharin solution (1 g/l) was paired to the injection of ethanol (0, 0.5, or 1 g/kg, i.p.), delivered immediately after removal of the saccharin bottle (conditioning phase). Subsequently, the choice between saccharin solution and water was offered for 18 consecutive daily 20-min sessions (postconditioning phase). Ethanol at 1g/kg produced a marked aversion to saccharin in sNP rats: The reduction in saccharin intake was already evident on the second day of the conditioning phase and lasted for 15 days of the postconditioning phase. In contrast, this dose of ethanol elicited a modest, if any, conditioned taste aversion in sP rats, although blood ethanol levels were comparable to those assessed in sNP rats. These results indicate the existence of a differential degree of aversion to the postingestional effects of ethanol between sP and sNP rats, and support the suggestion that it may contribute, at least in part, to the opposite preference for and consumption of ethanol monitored in these rat lines.

Reduced TH-immunoreactive fibers in the limbic system of Sardinian alcohol-preferring rats

The mesolimbic dopamine (DA) system has long been known to be involved in reward behaviors. As with other substances of abuse, it has been extensively reported that ethanol influences the dopaminergic system. The present study examined whether selectively bred Sardinian-alcohol-preferring (sP) and Sardinian alcohol non-preferring rats (sNP), differ in the DA innervation in structures of the forebrain that are related to rewarding behaviors. To this aim, we performed an immunohistochemistry study with an antibody raised against tyrosine hydroxylase (TH), the rate-limited step enzyme in the biosynthesis of monoamines. The TH-positive innervation density was found to be significantly lower in the cingulate cortex and in the shell of the nucleus accumbens of the sP when compared with the sNP and unselected Wistar rats. These anatomical structures both cluster in the medial aspect of the mesolimbic system. No differences in other major DA brain regions, such as the nigro-striatal pathway were found. The analysis of cell-body area revealed no differences between sP, sNP and Wistar rats in the ventral tegmental area and substantia nigra (pars compacta and reticulata) and the density of the TH-positive fibers was not different in the caudate-putamen. These results indicate a selective reduction of terminal innervation in the medial portion of the mesocorticolimbic DA system in sP rats and suggest that the latter may consume larger amounts of ethanol, when compared with sNP rats, to compensate for the deficiency of dopamine to produce an adequate level of reward.

Low responsiveness to agents evoking 5-HT 2 receptor-mediated behaviors in Sardinian alcohol-preferring rats

The selective NK3 tachykinin agonist senktide evokes in rodents 5-HT mediated behaviors, including 5-HT 2 receptor-mediated wet dog shakes (WDS) and head shakes (HS). It was observed previously that genetically selected Sardinian alcohol-preferring (sP) rats show a small number of WDS and HS following intracerebroventricular (ICV) injection of senktide. The present study was aimed at confirming these observations and at providing information on the reasons accounting for the anomalous response of sP rats. Senktide (500–2000 ng/rat, ICV) produced a much lower number of WDS and HS in sP rats than in nonselected Wistar (nsW) rats. Both behaviors were suppressed by the 5-HT 2 antagonist ritanserin (1 mg/kg, subcutaneously), confirming that 5-HT 2 receptors mediate the response. HS induced by the ICV injection of 5-HT agonists endowed with marked activity at 5-HT 2 receptors, such as quipazine (1500–6000 ng/rat) or DOI (500–3500 ng/rat), were much less pronounced in sP rats than in nsW rats. Moreover, WDS following peripheral injection of 5-hydroxytryptophan, 25–100 mg/kg and carbidopa, 12.5 mg/kg, were less intense in sP and in ethanol-naive sP rats than in nsW and in Sardinian alcohol-nonpreferring rats. These findings suggest that sP rats have an inherent different regulation of central 5-HT 2 mechanisms.