Paula J. Bice

Genomic screen for QTLs underlying alcohol consumption in the P and NP rat lines

Abstract. Selective breeding for voluntary alcohol consumption was utilized to establish the alcohol-preferring (P) and alcohol-nonpreferring (NP) rat lines. Inbreeding was initiated after 30 generations of selection and, after 19 generations of inbreeding, 384 F2 intercross progeny were created to identify quantitative trait loci (QTLs) influencing alcohol consumption. We had reported previously a QTL on Chromosome (Chr) 4; additional markers genotyped on Chr 4 have increased the maximum lod score from 8.6 to 9.2. This QTL acts in an additive fashion and continues to account for approximately 11% of the phenotypic variability. The 95% confidence interval is 12.5 cM and includes the candidate gene, neuropeptide Y. Subsequent to the identification of the QTL on Chr 4, a genome scan was completed to identify additional QTLs influencing alcohol consumption. A lod score of 2.5 was obtained on Chr 3, syntenic to a region previously reported for alcohol preference in mice. Analysis of Chr 8 produced a lod score of 2.2 near the dopamine D2 and serotonin 1b receptors, which have been previously reported as candidate genes for alcohol preference. Evidence for linkage to alcohol consumption was not found on any other chromosome. It therefore appears likely that, in addition to the QTL on Chr 4, multiple loci of small to moderate effect, such as those on Chrs 3 and 8, underlie the difference in alcohol consumption in the P/NP lines.

Candidate genes and their regulatory elements: alcohol preference and tolerance.

QTL analysis of behavioral traits and mouse brain gene expression studies were combined to identify candidate genes involved in the traits of alcohol preference and acute functional alcohol tolerance. The systematic application of normalization and statistical analysis of differential gene expression, behavioral and expression QTL location, and informatics methodologies resulted in identification of 8 candidate genes for the trait of alcohol preference and 22 candidate genes for acute functional tolerance. Pathway analysis, combined with clustering by ontology, indicated the importance of transcriptional regulation and DNA and protein binding elements in the acute functional tolerance trait, and protein kinases and intracellular signal transduction elements in the alcohol preference trait. A rudimentary search for transcription control elements that could indicate coregulation of the panels of candidate genes produced modest results, implicating SMAD-3 in the regulation of four of the eight candidate genes for alcohol preference. However, the realization of the many caveats related to transcription factor binding site analysis, and attempts to correlate between transcription factor binding and function, forestalled any definitive global analysis of transcriptional control of differentially expressed candidate genes.

Identification of quantitative trait loci influencing alcohol consumption in the high alcohol drinking and low alcohol drinking rat lines.

Selective breeding has been employed to develop high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rat lines from the heterogeneous N/Nih rat. Within-family selection and a rotational breeding design were used to discourage inbreeding (Li et al, 1993). To identify quantitative trait loci (QTLs) contributing to alcohol consumption, reciprocal HAD and LAD matings in conjunction with F1 intercrosses were used to create 459 F2 progeny. Using selective genotyping of 151 F2 progeny with extreme alcohol consumption scores and a novel least squares method developed by Haley et al (1994), five chromosomal regions (1,5,10,12, and 16) were identified with lod scores greater than 2.0. Genotyping of the entire sample of 459 F2 progeny produced maximum lod scores of 3.5 on chromosome 5, 2.4 on chromosome 10, 4.7 on chromosome 12 and 2.9 on chromosome 16. The evidence of linkage to chromosome 1 diminished substantially to a maximum lod score of 0.5 when all F2 progeny were genotyped. This study is the first genome-wide study for QTLs underlying alcohol consumption that has employed noninbred lines. Further localization of these QTLs will likely provide insight and candidate genes for the study of human alcoholism.

Similarity of taste reactivity responses to alcohol and sucrose mixtures in rats

The orofacial responses of rats following infusion of taste solutions were examined in two experiments. In the first experiment, naive rats were presented with a 6% alcohol solution and three sucrose mixtures (sucrose combined with quinine hydrochloride, hydrochloric acid, and sodium chloride, respectively) on separate trials and the resulting taste reactivity was examined. The only difference among the solutions was that alcohol elicited a significantly larger number of aversive responses (e.g., gapes, passive drips) than the sucrose mixtures. In the second experiment, naive rats were trained to avoid 6% alcohol using standard conditioned aversion procedures; rats were then tested for reactivity to the three sucrose mixtures and the alcohol solution. With the alcohol solution, trained rats displayed significantly fewer ingestive responses and significantly more aversive responses than control rats. The response of trained rats to the sucrose + quinine solution was similar to that of alcohol: fewer ingestive responses and more aversive responses than control rats. The number of aversive responses to the alcohol and the sucrose + quinine mixture by the trained rats did not differ significantly. Reactivity to the sucrose + hydrochloric acid and sucrose + sodium chloride solutions did not differ between trained rats and control rats. The results suggest that a sucrose + quinine solution has a perceived taste (as revealed by elicited orofacial reflexes) similar to alcohol and that the sucrose mixture is avoided by rats with alcohol aversions because it is unpalatable.

Developmental alterations of ethanol sensitivity in selectively bred high and low alcohol sensitive rats

Initial sensitivity and acute tolerance to ethanol have been implicated as risk factors in the development of alcoholism in humans. These behaviors were investigated in rats selectively bred for differences in hypnotic sensitivity following their first dose of ethanol in two different experiments. In Experiment 1, developmental profiles of the association between initial sensitivity and acute tolerance induced by a single exposure to ethanol were examined using male and female high, low, and control alcohol sensitive (HAS, LAS, and CAS) rats. Dose-response curves were constructed for duration of the loss of the righting reflex and for blood ethanol concentration (BEC) at the regain of the righting reflex. Animals were tested with a single ethanol dose ranging from 1.5 to 5.0 g/kg at either 15, 25, 40, 70, 120, or 180 days of age (DOA). For each group, acute tolerance to ethanol was estimated by the slope of the regression line using dose of ethanol and mean BEC at regain. In general, all rat lines showed an increase in hypnotic sensitivity to ethanol with age. To a large degree, the lower sensitivity observed in 15 and 25 DOA HAS and LAS rats was associated with an increase in the development of acute ethanol tolerance relative to older rats. Divergence of the LAS and CAS lines was evident by 25 DOA and remained stable with advancing age. However, HAS rats did not differ significantly from CAS rats until 40 DOA, after which the magnitude of the difference continued to increase with age. In Experiment 2, rats were treated with alcohol at 25, 70, or 180 DOA. Rats at 70 or 180 DOA required less ethanol to disrupt their motor coordination on a rotating dowel (rotarod). Blood ethanol levels were determined at the loss and subsequent regain of the ability to negotiate the rotarod. Total duration of inability to negotiate the rotarod also was recorded. HAS rats were less able to remain on a rotarod while under the influence of alcohol relative to LAS and CAS rats regardless of age. However, no evidence of acute tolerance was observed in this experiment and, in fact, there was evidence of reverse tolerance in that all animals had lower BEC values at regain of ability than they did at loss.

Drd2 expression in the high alcohol-preferring and low alcohol-preferring mice

The high alcohol-preferring (HAP) and low alcohol-preferring (LAP) mice were selectively bred for differences in alcohol preference and consumption. Recently, a large-effect QTL was identified on chromosome 9. The peak for this QTL is near the Drd2 (dopamine receptor 2) locus. The present study examined Drd2 mRNA expression differences between the HAP1 and LAP1 mice in brain regions important in the dopaminergic-reward pathway, including the nucleus accumbens, hippocampus, amygdala, and septum. Results show that alcohol-naïve HAP1 mice exhibited lower levels of Drd2 mRNA expression in the nucleus accumbens and the hippocampus compared to LAP1 mice. No differences were found in the amygdala or septum. To determine if a sequence difference might underlie the expression difference, the Drd2 cDNA was sequenced in each line and one single nucleotide polymorphism (SNP) was identified in the 3′ UTR. Both HAP and LAP 3′ UTR were cloned in the luc-pGL3-promoter-luc vector. The polymorphism in the Drd2 3′ UTR was assessed to determine its functional significance in modulating expression. In vitro expression analysis using neuroblastoma SK-N-SH cells resulted in a significant decrease in expression of the HAP 3′ UTR luc construct compared with the LAP 3′ UTR construct. This decreased expression is consistent with lower levels of Drd2 expression in the nucleus accumbens and the hippocampus as evidenced by qRT-PCR. These results suggest that the SNP may play a role in the differential expression of Drd2 between the HAP and LAP mice and that the polymorphism in Drd2 may contribute to alcohol preference.

Mapping of QTL influencing saccharin consumption in the selectively bred alcohol-preferring and -nonpreferring rat lines.

The inbred preferring (iP) and nonpreferring (iNP) rat strains were derived from the selectively bred alcohol-preferring (P) and alcohol-nonpreferring (NP) lines. Previously, 381 iP × iNP F2 progeny were generated to identify quantitative trait loci (QTLs) influencing alcohol consumption and preference. Saccharin consumption (ml/48 h) and saccharin intake (ml/kg/day) were also measured in the F2 sample and were significantly correlated with both alcohol consumption and preference (all r ≥ .20, p < .0001), suggesting that there might be some QTLs influencing both saccharin and alcohol phenotypes. We have performed a genome screen using F2 animals with extreme saccharin or alcohol consumption to identify QTLs contributing to saccharin-related phenotypes. Lod scores greater than 2.0 were found on chromosomes 3, 16 and 18 in this sample. Additional genotyping was performed in these regions in the full sample of 381 F2 progeny to further characterize these putative QTLs. On chromosome 3, the maximum lod score in the full sample was 2.7 with saccharin consumption. This QTL appears to overlap with a QTL identified for alcohol consumption in the iP and iNP lines and has pleiotropic effects on both phenotypes. Interestingly, this region of rat chromosome 3 is syntenic with mouse chromosome 2, where a QTL influencing alcohol preference has been previously reported. The QTL on chromosome 16 has a maximum lod score of 4.0 with saccharin intake and 2.6 with saccharin consumption. The QTL on chromosome 18 has a maximum lod score of 2.7 with saccharin consumption. Taken together, these data provide the first results of a genome screen for QTLs contributing to saccharin phenotypes in the rat.

Genomewide SNP screen to detect quantitative trait loci for alcohol preference in the high alcohol preferring and low alcohol preferring mice

BACKGROUND The high and low alcohol preferring (HAP1 and LAP1) mouse lines were selectively bred for differences in alcohol intake. The HAP1 and LAP1 mice are essentially noninbred lines that originated from the outbred colony of HS/Ibg mice, a heterogeneous stock developed from intercrossing 8 inbred strains of mice. METHODS A total of 867 informative SNPs were genotyped in 989 HAP1 x LAP1 F2, 68 F1s, 14 parents (6 LAP1, 8 HAP1), as well as the 8 inbred strains of mice crossed to generate the HS/Ibg colony. Multipoint genome wide analyses were performed to simultaneously detect linked QTLs and also fine map these regions using the ancestral haplotypes. RESULTS QTL analysis detected significant evidence of association on 4 chromosomes: 1, 3, 5, and 9. The region on chromosome 9 was previously found linked in a subset of these F2 animals using a whole genome microsatellite screen. CONCLUSIONS We have detected strong evidence of association to multiple chromosomal regions in the mouse. Several of these regions include candidate genes previously associated with alcohol dependence in humans or other animal models.

Evaluating the palatability of alcohol for rats with measures of taste reactivity, consumption, and lick rate

In two experiments, rats were presented with water and six concentrations of alcohol (0.5%, 3%, 6%, 9%, 12%, and 15%, v/v) under conditions of mild fluid deprivation. Their responses were measured using taste reactivity (fluids infused directly into the mouth), consumption, and lick rate (both in a voluntary drinking situation). Results in both experiments showed that the number of overall ingestive responses was relatively high and consistent across all alcohol concentrations and water; aversive responding was low for all solutions. During one-bottle, 10-min tests, rats consumed the three lowest concentrations of alcohol (0.5%, 3%, and 6%) and water at an equal level. There was an abrupt drop in the amount of alcohol consumed at 9% and a continued decrease at the two highest concentrations. Lick rates for alcohol (measured only in Experiment 2) fell into three general patterns: 0.5%, 3%, and 6% produced almost identical, negatively accelerating curves consistently above that of water which was linear; lick rates for 9% and 12% rose initially but, at approximately 2 min, became flat; the 15% solution produced a low lick rate throughout the 5-min period. There were significant correlations between lick rate and amount consumed but, contrary to expectations, no significant correlations were found between taste reactivity and the other two measures (consumption and lick rate). These results suggest that taste reactivity to alcohol solutions may be reflective of processes different from those that regulate licking behavior or actual consumption.

Fine mapping and expression of candidate genes within the chromosome 10 QTL region of the high and low alcohol-drinking rats

The high and low alcohol-drinking (HAD and LAD) rats were selectively bred for differences in alcohol intake. The HAD/LAD rats originated from the N/Nih heterogeneous stock developed from intercrossing eight inbred rat strains. The HAD×LAD F2 were genotyped, and a powerful analytical approach, using ancestral recombination and F2 recombination, was used to narrow a quantitative trait loci (QTL) for alcohol drinking to a 2-cM region on distal chromosome 10 that was in common in the HAD1/LAD1 and HAD2/LAD2 analyses. Quantitative real-time PCR was used to examine mRNA expression of six candidate genes (Crebbp, Trap1, Gnptg, Clcn7, Fahd1, and Mapk8ip3) located within the narrowed QTL region in the HAD1/LAD1 rats. Expression was examined in five brain regions, including the nucleus accumbens, amygdala, caudate putamen, hippocampus, and prefrontal cortex. All six genes showed differential expression in at least one brain region. Of the genes tested in this study, Crebbp and Mapk8ip3 may be the most promising candidates with regard to alcohol drinking.