Systems genetics analysis of the LXS recombinant inbred mouse strains:Genetic and molecular insights into acute ethanol tolerance.

We have been using the Inbred Long- and Short-Sleep mouse strains (ILS, ISS) and a recombinant inbred panel derived from them, the LXS, to investigate the genetic underpinnings of acute ethanol tolerance which is considered to be a risk factor for alcohol use disorders (AUDs). Here, we have used RNA-seq to examine the transcriptome of whole brain in 40 of the LXS strains 8 hours after a saline or ethanol "pretreatment" as in previous behavioral studies. Approximately 1/3 of the 14,184 expressed genes were significantly heritable and many were unique to the pretreatment. Several thousand cis- and trans-eQTLs were mapped; a portion of these also were unique to pretreatment. Ethanol pretreatment caused differential expression (DE) of 1,230 genes. Gene Ontology (GO) enrichment analysis suggested involvement in numerous biological processes including astrocyte differentiation, histone acetylation, mRNA splicing, and neuron projection development. Genetic correlation analysis identified hundreds of genes that were correlated to the behaviors. GO analysis indicated that these genes are involved in gene expression, chromosome organization, and protein transport, among others. The expression profiles of the DE genes and genes correlated to AFT in the ethanol pretreatment group (AFT-Et) were found to be similar to profiles of HDAC inhibitors. Hdac1, a cis-regulated gene that is located at the peak of a previously mapped QTL for AFT-Et, was correlated to 437 genes, most of which were also correlated to AFT-Et. GO analysis of these genes identified several enriched biological process terms including neuron-neuron synaptic transmission and potassium transport. In summary, the results suggest widespread genetic effects on gene expression, including effects that are pretreatment-specific. A number of candidate genes and biological functions were identified that could be mediating the behavioral responses. The most prominent of these was Hdac1 which may be regulating genes associated with glutamatergic signaling and potassium conductance.

High-Resolution Maps of Mouse Reference Populations.

Genetic reference panels are widely used to map complex, quantitative traits in model organisms. We have generated new high-resolution genetic maps of 259 mouse inbred strains from recombinant inbred strain panels (C57BL/6J × DBA/2J, ILS/IbgTejJ × ISS/IbgTejJ, and C57BL/6J × A/J) and chromosome substitution strain panels (C57BL/6J-Chr#, C57BL/6J-Chr#, and C57BL/6J-Chr#). We genotyped all samples using the Affymetrix Mouse Diversity Array with an average intermarker spacing of 4.3 kb. The new genetic maps provide increased precision in the localization of recombination breakpoints compared to the previous maps. Although the strains were presumed to be fully inbred, we found residual heterozygosity in 40% of individual mice from five of the six panels. We also identified de novo deletions and duplications, in homozygous or heterozygous state, ranging in size from 21 kb to 8.4 Mb. Almost two-thirds (46 out of 76) of these deletions overlap exons of protein coding genes and may have phenotypic consequences. Twenty-nine putative gene conversions were identified in the chromosome substitution strains. We find that gene conversions are more likely to occur in regions where the homologous chromosomes are more similar. The raw genotyping data and genetic maps of these strain panels are available at http://churchill-lab.jax.org/website/MDA.

Genome characterization of the selected long- and short-sleep mouse lines.

The Inbred Long- and Short-Sleep (ILS, ISS) mouse lines were selected for differences in acute ethanol sensitivity using the loss of righting response (LORR) as the selection trait. The lines show an over tenfold difference in LORR and, along with a recombinant inbred panel derived from them (the LXS), have been widely used to dissect the genetic underpinnings of acute ethanol sensitivity. Here we have sequenced the genomes of the ILS and ISS to investigate the DNA variants that contribute to their sensitivity difference. We identified ~2.7 million high-confidence SNPs and small indels and ~7000 structural variants between the lines; variants were found to occur in 6382 annotated genes. Using a hidden Markov model, we were able to reconstruct the genome-wide ancestry patterns of the eight inbred progenitor strains from which the ILS and ISS were derived, and found that quantitative trait loci that have been mapped for LORR were slightly enriched for DNA variants. Finally, by mapping and quantifying RNA-seq reads from the ILS and ISS to their strain-specific genomes rather than to the reference genome, we found a substantial improvement in a differential expression analysis between the lines. This work will help in identifying and characterizing the DNA sequence variants that contribute to the difference in ethanol sensitivity between the ILS and ISS and will also aid in accurate quantification of RNA-seq data generated from the LXS RIs.

A Community-Based Exercise and Support Group Program Improves Quality of Life in African-American Breast Cancer Survivors: A Quantitative and Qualitative Analysis.

African-American (AA) breast cancer (BCa) survivors have higher mortality rates, more comorbidities and are less likely to meet national physical activity guidelines after diagnosis compared to Caucasian BCa survivors. We previously reported that a 20-week resistance exercise intervention coupled with a support group and home walking program, conducted using facilities and personnel at a community cancer support center, in Stage I-III AA BCa survivors improved strength, fitness and circulating C-peptide levels. Here, we report our findings on changes in quality of life (QoL) and other behavioral measures associated with this 20-week intervention and, discuss findings from a qualitative analysis of semi-structured patient interviews. We found a clinically relevant improvement in QoL using the Functional Assessment of Cancer Therapy for Breast Cancer (FACT-B) (Baseline, B: 101.1 ± 21.5; End-of-Intervention, EOI: 108.5 ± 21.6; p = 0.05) and, a significant decrease in depression using the Beck Depression Inventory-II (B: 11.9 ± 8.1; EOI: 9.0 ± 5.5; p = 0.03). Our analysis of the patient interviews support improvements in these behavioral measures in that participants stated that they "feel better", were "more motivated" and "uplifted" after the program. The patient interviews also provided insights to the primary motivators (e.g., social support, improvements in strength and function, weight loss) and barriers (e.g., family and health issues) in adhering to the program and provided suggestions for improving the program (e.g., incorporating nutritional and treatment related side-effect discussions). Our results suggest that community-based lifestyle interventions may improve QoL and depression in AA BCa survivors and lend insights for improving future programs.

Quantitative trait locus mapping of acute functional tolerance in the LXS recombinant inbred strains.

BACKGROUND: We previously reported that acute functional tolerance (AFT) to the hypnotic effects of alcohol was significantly correlated with drinking in the dark (DID) in the LXS recombinant inbred panel, but only in mice that had been pretreated with alcohol. Here, we have conducted quantitative trait locus (QTL) mapping for AFT. DNA sequencing of the progenitor ILS and ISS strains and microarray analyses were also conducted to identify candidate genes and functional correlates. METHODS: LXS mice were given either saline or alcohol (5 g/kg) on day 1 and then tested for loss of righting reflex AFT on day 2. QTLs were mapped using standard procedures. Two microarray analyses from brain were conducted: (i) naïve LXS mice and (ii) an alcohol treatment time course in the ILS and ISS. The full genomes of the ILS and ISS were sequenced to a depth of approximately 30×. RESULTS: A significant QTL for AFT in the alcohol pretreatment group was mapped to distal chromosome 4; numerous suggestive QTLs were also mapped. Preference drinking and DID have previously been mapped to the chromosome 4 locus. The credible interval of the significant chromosome 4 QTL spanned 23 Mb and included 716 annotated genes of which 150 had at least 1 nonsynonymous single nucleotide polymorphism or small indel that differed between the ILS and ISS; expression of 48 of the genes was cis-regulated. Enrichment analysis indicated broad functional categories underlying AFT, including proteolysis, transcription regulation, chromatin modification, protein kinase activity, and apoptosis. CONCLUSIONS: The chromosome 4 QTL is a key region containing possibly pleiotropic genes for AFT and drinking behavior. Given that the region contains many viable candidates and a large number of the genes in the interval fall into 1 or more of the enriched functional categories, we postulate that many genes of varying effect size contribute to the observed QTL effect.

Influence of sex on genetic regulation of "drinking in the dark" alcohol consumption.

The ILSXISS (LXS) recombinant inbred (RI) panel of mice is a valuable resource for genetic mapping studies of complex traits, due to its genetic diversity and large number of strains. Male and female mice from this panel were used to investigate genetic influences on alcohol consumption in the "drinking in the dark" (DID) model. Male mice (38 strains) and female mice (36 strains) were given access to 20% ethanol during the early phase of their circadian dark cycle for four consecutive days. The first principal component of alcohol consumption measures on days 2, 3, and 4 was used as a phenotype (DID phenotype) to calculate QTLs, using a SNP marker set for the LXS RI panel. Five QTLs were identified, three of which included a significant genotype by sex interaction, i.e., a significant genotype effect in males and not females. To investigate candidate genes associated with the DID phenotype, data from brain microarray analysis (Affymetrix Mouse Exon 1.0 ST Arrays) of male LXS RI strains were combined with RNA-Seq data (mouse brain transcriptome reconstruction) from the parental ILS and ISS strains in order to identify expressed mouse brain transcripts. Candidate genes were determined based on common eQTL and DID phenotype QTL regions and correlation of transcript expression levels with the DID phenotype. The resulting candidate genes (in particular, Arntl/Bmal1) focused attention on the influence of circadian regulation on the variation in the DID phenotype in this population of mice.

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.

Genetic studies of acute tolerance, rapid tolerance, and drinking in the dark in the LXS recombinant inbred strains.

BACKGROUND: We hypothesized that rapid tolerance (1-day tolerance) for the duration of the loss of righting reflex ("sleep time" [ST]) was mediated by an increase in acute functional tolerance (AFT). We also hypothesized that increased AFT would correspond to increased drinking. These questions were addressed using the LXS recombinant inbred mouse strain panel. METHODS: Mice were given a pretreatment dose of either saline or 5 g/kg alcohol on day 1. On day 2, mice were tested for ST (4.1 g/kg) using a method with which it is possible to accurately assess AFT. Genetic correlation analysis was conducted among the ST-related variables and also with "drinking in the dark" (DID) which was previously measured by Saba and colleagues (2011). RESULTS: Saline-pretreated mice showed a continuous distribution of ST ranging from ~40 minutes to over 3 hours. Of the 43 strains tested, 9 showed significantly decreased ST after alcohol pretreatment, while in 3 strains, ST was significantly increased. AFT scores ranged from 0 to over 200 mg% in the saline group, and in the alcohol group, 8 strains showed a significant increase in AFT and 2 strains showed significant decrease in AFT. In the saline group, AFT was significantly correlated with ST (r = -0.47), but not in the alcohol group (r = -0.22). DID was significantly correlated with only AFT in the alcohol pretreated group (r = 0.64). CONCLUSIONS: The results suggest that AFT is an important component of the overall ST response, but that the alcohol pretreatment-induced change in AFT does not contribute to rapid ST tolerance. The significant correlation between DID and AFT in the alcohol group suggests that AFT may be a more relevant predictor of drinking behavior than the static measurement of ST. Moreover, preexposure to alcohol seems to change AFT in a way that makes it an even stronger predictor of drinking behavior.

Expression of alcoholism-relevant genes in the liver are differently correlated to different parts of the brain.

The purpose of this study is to investigate whether expression profiles of alcoholism-relevant genes in different parts of the brain are correlated differently with those in the liver. Four experiments were conducted. First, we used gene expression profiles from five parts of the brain (striatum, prefrontal cortex, nucleus accumbens, hippocampus, and cerebellum) and from liver in a population of recombinant inbred mouse strains to examine the expression association of 10 alcoholism-relevant genes. Second, we conducted the same association analysis between brain structures and the lung. Third, using five randomly selected, nonalcoholism-relevant genes, we conducted the association analysis between brain and liver. Finally, we compared the expression of 10 alcoholism-relevant genes in hippocampus and cerebellum between an alcohol preference strain and a wild-type control. We observed a difference in correlation patterns in expression levels of 10 alcoholism-relevant genes between different parts of the brain with those of liver. We then examined the association of gene expression between alcohol dehydrogenases (Adh1, Adh2, Adh5, and Adh7) and different parts of the brain. The results were similar to those of the 10 genes. Then, we found that the association of those genes between brain structures and lung was different from that of liver. Next, we found that the association patterns of five alcoholism-nonrelevant genes were different from those of 10 alcoholism-relevant genes. Finally, we found that the expression level of 10 alcohol-relevant genes is influenced more in hippocampus than in cerebellum in the alcohol preference strain. Our results show that the expression of alcoholism-relevant genes in liver is differently associated with the expression of genes in different parts of the brain. Because different structural changes in different parts of the brain in alcoholism have been reported, it is important to investigate whether those structural differences in the brains of those with alcoholism are due to the difference in the associations of gene expression between genes in liver and in different parts of the brain.

A systems genetic analysis of alcohol drinking by mice, rats and men: influence of brain GABAergic transmission.

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'.

Genetical genomic analysis of complex phenotypes using the PhenoGen website.

Our laboratory has developed an online interactive resource called PhenoGen ( http://phenogen.ucdenver.edu ) which provides an archive of brain and other organ gene expression data from a panel of 20 common inbred mouse strains, and three recombinant inbred (RI) panels (two mouse and one rat). DNA microarray data can also be uploaded to the site where numerous analytical tools can be implemented. An important advantage to the archived data is that each array represents data from a single animal and each strain was sampled 4-7 times, providing an estimate of genetic variance (heritability) of individual transcript levels. These panels also allow genetic mapping of expression QTLs. Overlap of eQTLs with phenotypic QTLs provides a powerful approach to candidate gene identification. These methods are briefly described here and we encourage the use of our site for both scientific discovery and as a teaching tool in quantitative genetics.

Using the Phenogen website for 'in silico' analysis of morphine-induced analgesia: identifying candidate genes.

The identification of genes that contribute to polygenic (complex) behavioral phenotypes is a key goal of current genetic research. One approach to this goal is to combine gene expression information with genetic information, i.e. to map chromosomal regions that regulate gene expression levels. This approach has been termed 'genetical genomics', and, when used in conjunction with the identification of genomic regions (QTLs) that regulate the complex physiological trait under investigation, provides a strong basis for candidate gene discovery. In this paper, we describe the implementation of the genetical genomic/phenotypic approach to identify candidate genes for sensitivity to the analgesic effect of morphine in BXD recombinant inbred mice. Our analysis was performed 'in silico', using an online interactive resource called PhenoGen (http://phenogen.ucdenver.edu). We describe in detail the use of this resource, which identified a set of candidate genes, some of whose products regulate the cellular localization and activity of the mu opiate receptor. The results demonstrate how PhenoGen can be used to identify a novel set of genes that can be further investigated for their potential role in pain, morphine analgesia and/or morphine tolerance.

Phenotype and genetics of progressive sensorineural hearing loss (Snhl1) in the LXS set of recombinant inbred strains of mice.

Progressive sensorineural hearing loss is the most common form of acquired hearing impairment in the human population. It is also highly prevalent in inbred strains of mice, providing an experimental avenue to systematically map genetic risk factors and to dissect the molecular pathways that orchestrate hearing in peripheral sensory hair cells. Therefore, we ascertained hearing function in the inbred long sleep (ILS) and inbred short sleep (ISS) strains. Using auditory-evoked brain stem response (ABR) and distortion product otoacoustic emission (DPOAE) measurements, we found that ISS mice developed a high-frequency hearing loss at twelve weeks of age that progressed to lower frequencies by 26 weeks of age in the presence of normal endocochlear potentials and unremarkable inner ear histology. ILS mice exhibited milder hearing loss, showing elevated thresholds and reduced DPOAEs at the higher frequencies by 26 weeks of age. To map the genetic variants that underlie this hearing loss we computed ABR thresholds of 63 recombinant inbred stains derived from the ISS and ILS founder strains. A single locus was linked to markers associated with ISS alleles on chromosome 10 with a highly significant logarithm of odds (LOD) score of 15.8. The 2-LOD confidence interval spans approximately 4 Megabases located at position 54-60 Mb. This locus, termed sensorineural hearing loss 1 (Snhl1), accounts for approximately 82% of the phenotypic variation. In summary, this study identifies a novel hearing loss locus on chromosome 10 and attests to the prevalence and genetic heterogeneity of progressive hearing loss in common mouse strains.

Differences in the urocortin 1 system between long-sleep and short-sleep mice.

There is evidence that the peptide urocortin 1 (Ucn1) may be involved in mediating some of the effects of ethanol. The purpose of the present study was to characterize Ucn1 immunoreactivity in mice selectively bred for either high or low sensitivity to ethanol-induced sedation, with additional differences in their response to ethanol-induced hypothermia. The brains of naïve male mice of the inbred long sleep/short sleep (ILS/ISS) selected lines were analyzed by immunohistochemistry. Significant differences were found between lines in the number of Ucn1-containing cells in the non-preganglionic Edinger-Westphal nucleus (npEW, the main source of Ucn1 in the brain); with the ISS mice having more cells. However, significant differences in the optical density of Ucn1 immunoreactivity in individual npEW cells and differences in cell area were also found between lines, with ILS mice having a greater density of Ucn1 per cell and having larger cells in the npEW. Importantly, the ILS mice also had a significantly greater number of Ucn1-positive terminal fibers than ISS mice in the lateral septum and the dorsal raphe nucleus, projection areas of Ucn1-containing neurons. These results suggest that the greater sensitivity of ILS than ISS mice to the hypothermic effects of ethanol could be mediated by stronger innervation of the dorsal raphe by Ucn1-containing fibers. In addition, these results lend further support to previous findings implicating Ucn1-containing projections from npEW to the dorsal raphe in ethanol-induced hypothermia.

Quantitative trait locus mapping for acute functional tolerance to ethanol in the L x S recombinant inbred panel.

BACKGROUND: Acute functional tolerance (AFT) develops shortly after ethanol administration, and is determined as the change in brain or blood ethanol concentration (BEC) measured at 2 behavioral or physiological endpoints. Acute functional tolerance studies in some rodent strains support a long-held hypothesis that more sensitive strains develop more within-session tolerance. We used the new, 74-strain L x S recombinant inbred (RI) panel, developed from inbred long-sleep (ILS) and inbred short-sleep (ISS) strains, to revisit this hypothesis and to map quantitative trait loci (QTLs) for AFT. We report replication of QTL regions reported by earlier studies of AFT and preliminary application of a coarse single nucleotide polymorphism map analysis to limit QTL intervals for subsequent candidate gene hypotheses. METHODS: Acute functional tolerance was assayed using a test of ataxia: loss and regain of balance on a stationary wooden dowel. Following an initial dose of 1.75 g/kg, BEC was measured at initial loss (BEC(0)) and regain of balance (BEC(1)). A second injection (2.0 g/kg) was administered and blood taken at the second regain of balance (BEC(2)). Acute functional tolerance was calculated as a difference score in 2 ways: (1) between BEC at the 2 successive regains of balance (AFT(1)), or (2) as the difference in BEC at final regain and at initial loss of balance (AFT(2)). We mapped QTLs for BEC(0), a measure of initial sensitivity, and both AFT scores. RESULTS: All 4 parental strains (LS, SS, ILS, and ISS) developed tolerance, replicating previous published reports. There were significant sex effects for 3 of these strains. The L x S panel showed a 128-fold range in tolerance, with a few strains showing negative tolerance (sensitization). The ISS surpassed the next highest RI strain by 55% and was more than 4 times greater than SS. Heritability estimates for both AFT measures were close to 0.25 for both sexes. One significant QTL accounting for approximately 18% of phenotypic variance (V(P)), on chromosome 12 (AFT(1)), and 1 suggestive QTL (16% V(P)), on chromosome 16 (AFT(2)), were identified. These QTLs replicated regions reported in other studies. A multiple QTL model incorporating the effects of all significant interacting QTLs was developed, explaining almost 60% of V(P). The chromosome 12 region was further investigated by haplotype analysis, which identified many nonpolymorphic regions within the confidence interval, and possible candidate genes in the polymorphic regions. CONCLUSIONS: Both SS and ISS developed greater AFT, assessed by both methods, than LS and ILS; this difference was significant in virtually all sex by strain comparisons. In the L x S RI, there was no correlation between initial sensitivity, measured by BEC at initial loss of balance, and either measure of AFT, on a stationary dowel. These results indicate that in this model system, initial sensitivity does not predict tolerance. Several QTLs for tolerance were identified; candidates in the narrowed chromosome 12 region, which has been reported in 2 other mapping studies, merit additional study.

QTL mapping for low-dose ethanol activation in the LXS recombinant inbred strains.

BACKGROUND: Most mouse quantitative trait loci (QTLs) for behavioral traits have been mapped using populations of mice derived from C57BL/6J (B6) and DBA/2J (D2). It is also important to identify QTLs for behavior in populations derived from other progenitors. We report results from QTL mapping for low-dose (ethanol) locomotor activation (LDA) using the recently developed LXS recombinant inbred (RI) strains, derived from Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS) progenitors. The LXS RI panel has additional genetic variation, and greater power due to a larger number of strains, compared with other RI panels and strain crosses. METHODS: Mice were tested using a 3-day protocol in which activity levels were monitored for 15 minutes each day. On day 1, baseline activity was recorded; on day 2, mice were injected with saline before testing; and on day 3, mice were injected with 1.8 g/kg ethanol and tested. RESULTS: Several suggestive QTLs were found, on chromosomes 2, 3, 4, 7, 8, 12, and 13; 3 of these QTLs were sex-specific. CONCLUSIONS: Two apparently novel LDA QTLs were identified, on chromosomes 4 and 8. The other QTLs appear to replicate previously identified LDA QTLs. These replicated QTLs will be pursued in subsequent studies designed to identify candidate genes.

Confirmation and fine mapping of ethanol sensitivity quantitative trait loci, and candidate gene testing in the LXS recombinant inbred mice.

In previous studies, we have mapped quantitative trait loci (QTLs) for hypnotic sensitivity to ethanol using a small recombinant inbred (RI) panel and a large F(2) backcross. Alcohol sensitivity is a major predictor of long-term risk for alcoholism. We remapped hypnotic sensitivity using a new set of 75 RI strains, the LXS, derived from Inbred Long Sleep and Inbred Short Sleep strains. We expected to improve mapping resolution in the QTL regions and to identify novel QTLs for loss of the righting reflex due to ethanol. We used three common mapping algorithms (R/qtl, QTL Cartographer, and WebQTL) to map QTLs in the LXS, and we compared the results. Most mapping studies use only a single algorithm, an approach that may result in failure to identify minor QTLs. We confirmed most of our previously reported QTLs, although one major QTL from earlier work (Lore2) failed to replicate, possibly because it represented multiple linked genes separated by recombination in the RI strains. We also report narrowed confidence intervals, based on mapping with a new genetic resource of more than 4000 polymorphic single-nucleotide polymorphism markers. These narrowed confidence intervals will facilitate candidate gene identification and assessment of overlap with human regions specifying risk for alcoholism. Finally, we present an approach for using these RI strains to assess evidence for candidate genes in the narrowed intervals, and we apply this method to a strong candidate, the serotonin transporter.

Mouse genetic models in alcohol research.

Animal models offer several advantages for the study of complex human disorders such as alcoholism. No animal model replicates all aspects of alcoholism but different components of the disorder can be investigated using various rodent models. In this article, we review a select subset of the most widely used mouse genetic models in alcohol research. Different genetically defined strains and stocks of mice are useful for genetic, physiologic, behavioral and pharmacological studies of this devastating disorder. In the past decade, numerous genomic regions associated with a tendency for various behavioral components of alcoholism have been identified; recent applications of new methods are shedding light on quantitative trait genes. Many of the underlying genes should be identified in the near future.

Expression profiling identifies novel candidate genes for ethanol sensitivity QTLs.

The Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS) mouse strains have a 16-fold difference in duration of loss of the righting response (LORR) following administration of a sedative dose of ethanol. Four quantitative trait loci (QTLs) have been mapped in these strains for this trait. Underlying each of these QTLs must be one or more genetic differences (polymorphisms in either gene coding or regulatory regions) influencing ethanol sensitivity. Because prior studies have tended to focus on differences in coding regions, genome-wide expression profiling in cerebellum was used here to identify candidate genes for regulatory region differences in these two strains. Fifteen differentially expressed genes were found that map to the QTL regions and polymorphisms were identified in the promoter regions of four of these genes by direct sequencing of ILS and ISS genomic DNA. Polymorphisms in the promoters of three of these genes, Slc22a4, Rassf2, and Tax1bp3, disrupt putative transcription factor binding sites. Slc22a4 and another candidate, Xrcc5, have human orthologs that map to genomic regions associated with human ethanol sensitivity in genetic linkage studies. These genes represent novel candidates for the LORR phenotype and provide new targets for future studies into the neuronal processes underlying ethanol sensitivity.

Norepinephrine transporter: a candidate gene for initial ethanol sensitivity in inbred long-sleep and short-sleep mice.

BACKGROUND: Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. METHODS: In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. RESULTS: We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. CONCLUSIONS: This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.