Model based heritability scores for high-throughput sequencing data.

BACKGROUND: Heritability of a phenotypic or molecular trait measures the proportion of variance that is attributable to genotypic variance. It is an important concept in breeding and genetics. Few methods are available for calculating heritability for traits derived from high-throughput sequencing. RESULTS: We propose several statistical models and different methods to compute and test a heritability measure for such data based on linear and generalized linear mixed effects models. We also provide methodology for hypothesis testing and interval estimation. Our analyses show that, among the methods, the negative binomial mixed model (NB-fit), compound Poisson mixed model (CP-fit), and the variance stabilizing transformed linear mixed model (VST) outperform the voom-transformed linear mixed model (voom). NB-fit and VST appear to be more robust than CP-fit for estimating and testing the heritability scores, while NB-fit is the most computationally expensive. CP-fit performed best in terms of the coverage of the confidence intervals. In addition, we applied the methods to both microRNA (miRNA) and messenger RNA (mRNA) sequencing datasets from a recombinant inbred mouse panel. We show that miRNA and mRNA expression can be a highly heritable molecular trait in mouse, and that some top heritable features coincide with expression quantitative trait loci. CONCLUSIONS: The models and methods we investigated in this manuscript is applicable and extendable to sequencing experiments where some biological replicates are available and the environmental variation is properly controlled. The CP-fit approach for assessing heritability was implemented for the first time to our knowledge. All the methods presented, as well as the generation of simulated sequencing data under either negative binomial or compound Poisson mixed models, are provided in the R package HeritSeq.

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.

The multiMiR R package and database: integration of microRNA-target interactions along with their disease and drug associations.

microRNAs (miRNAs) regulate expression by promoting degradation or repressing translation of target transcripts. miRNA target sites have been catalogued in databases based on experimental validation and computational prediction using various algorithms. Several online resources provide collections of multiple databases but need to be imported into other software, such as R, for processing, tabulation, graphing and computation. Currently available miRNA target site packages in R are limited in the number of databases, types of databases and flexibility. We present multiMiR, a new miRNA-target interaction R package and database, which includes several novel features not available in existing R packages: (i) compilation of nearly 50 million records in human and mouse from 14 different databases, more than any other collection; (ii) expansion of databases to those based on disease annotation and drug microRNAresponse, in addition to many experimental and computational databases; and (iii) user-defined cutoffs for predicted binding strength to provide the most confident selection. Case studies are reported on various biomedical applications including mouse models of alcohol consumption, studies of chronic obstructive pulmonary disease in human subjects, and human cell line models of bladder cancer metastasis. We also demonstrate how multiMiR was used to generate testable hypotheses that were pursued experimentally.

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.

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 effects of ATP1A2 in familial hemiplegic migraine type II and animal models.

Na(+)/K(+)-ATPase alpha 2 (Atp1a2) is an integral plasma membrane protein belonging to the P-type ATPase family that is responsible for maintaining the sodium (Na(+)) and potassium (K(+)) gradients across cellular membranes with hydrolysis of ATP. Atp1a2 contains two subunits, alpha and beta, with each having various isoforms and differential tissue distribution. In humans, mutations in ATP1A2 are associated with a rare form of hereditary migraines with aura known as familial hemiplegic migraine type II. Genetic studies in mice have revealed other neurological effects of Atp1a2 in mice including anxiety, fear, and learning and motor function disorders. This paper reviews the recent findings in the literature concerning Atp1a2.

Alcohol inhibition of the NMDA receptor function, long-term potentiation, and fear learning requires striatal-enriched protein tyrosine phosphatase.

Alcohol's deleterious effects on memory are well known. Acute alcohol-induced memory loss is thought to occur via inhibition of NMDA receptor (NMDAR)-dependent long-term potentiation in the hippocampus. We reported previously that ethanol inhibition of NMDAR function and long-term potentiation is correlated with a reduction in the phosphorylation of Tyr(1472) on the NR2B subunit and ethanol's inhibition of the NMDAR field excitatory postsynaptic potential was attenuated by a broad spectrum tyrosine phosphatase inhibitor. These data suggested that ethanol's inhibitory effect may involve protein tyrosine phosphatases. Here we demonstrate that the loss of striatal-enriched protein tyrosine phosphatase (STEP) renders NMDAR function, phosphorylation, and long-term potentiation, as well as fear conditioning, less sensitive to ethanol inhibition. Moreover, the ethanol inhibition was "rescued" when the active STEP protein was reintroduced into the cells. Taken together, our data suggest that STEP contributes to ethanol inhibition of NMDAR function via dephosphorylation of tyrosine sites on NR2B receptors and lend support to the hypothesis that STEP may be required for ethanol's amnesic effects.

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.

Genetic Modifiers of Oral Nicotine Consumption in Chrna5 Null Mutant Mice.

The gene CHRNA5 is strongly associated with the level of nicotine consumption in humans and manipulation of the expression or function of Chrna5 similarly alters nicotine consumption in rodents. In both humans and rodents, reduced or complete loss of function of Chrna5 leads to increased nicotine consumption. However, the mechanism through which decreased function of Chrna5 increases nicotine intake is not well-understood. Toward a better understanding of how loss of function of Chrna5 increases nicotine consumption, we have initiated efforts to identify genetic modifiers of Chrna5 deletion-dependent oral nicotine consumption in mice. For this, we introgressed the Chrna5 knockout (KO) mutation onto a panel of C57BL/6J-Chr#A/J/NAJ chromosome substitution strains (CSS) and measured oral nicotine consumption in 18 CSS and C57BL/6 (B6) mice homozygous for the Chrna5 KO allele as well as their Chrna5 wild type littermates. As expected, nicotine consumption was significantly increased in Chrna5 KO mice relative to Chrna5 wildtype mice on a B6 background. Among the CSS homozygous for the Chrna5 KO allele, several exhibited altered nicotine consumption relative to B6 Chrna5 KO mice. Sex-independent modifiers were detected in CSS possessing A/J chromosomes 5 and 11 and a male-specific modifier was found on chromosome 15. In all cases nicotine consumption was reduced in the CSS Chrna5 KO mice relative to B6 Chrna5 KO mice and consumption in the CSS KO mice was indistinguishable from their wild type littermates. Nicotine consumption was also reduced in both Chrna5 KO and wildtype CSS mice possessing A/J chromosome 1 and increased in both KO and wild type chromosome 17 CSS relative to KO and wild type B6 mice. These results demonstrate the presence of several genetic modifiers of nicotine consumption in Chrna5 KO mice as well as identify loci that may affect nicotine consumption independent of Chrna5 genotype. Identification of the genes that underlie the altered nicotine consumption may provide novel insight into the mechanism through which Chrna5 deletion increases nicotine consumption and, more generally, a better appreciation of the neurobiology of nicotine intake.

Epistatic interactions govern chemically-induced lung tumor susceptibility and Kras mutation site in murine C57BL/6J-ChrA/J chromosome substitution strains.

Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#(A/J) strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6(A/J) mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A-->T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A-->G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.

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.

The Acute Influence of Acid Suppression with Esomeprazole on Gastrointestinal Microbiota and Brain Gene Expression Profiles in a Murine Model of Restraint Stress.

The central nervous system (CNS) and gastrointestinal tract (GIT) are linked through neuro-endocrine and humoral pathways. Critically ill patients suffer severe physical and emotional stress and frequently receive acid suppressants; however, stress and acid suppression may alter GIT microbiota. This study evaluated the effects of acid suppression on the GIT microbiota and genome-wide expression of brain-specific genes in a murine model of restraint stress. Twenty-four male C57BL/6J mice were randomly assigned to three days of restraint stress by hypothermic immobilization or control environment for three hours daily and either esomeprazole 2 mg/kg or saline by intraperitoneal injection daily. Bacterial communities associated with the stomach, ileum, cecum, and mid-colon were determined by broad-range 16S rRNA gene sequencing, while RNA-sequencing assessed mRNA expression in the hippocampus. Both stress (p < 0.001) and esomeprazole (p = 0.006) had significant, independent effects on the composition of stomach microbiota. Stress had no impact on the hippocampus but the addition of esomeprazole induced differential expression of 124 genes, many of which are involved in cognitive and behavior pathways. Gene expression was correlated with the abundances of multiple microbial families. Acute stress has region-specific effects on the distribution of GIT commensal bacteria which is heightened with acid suppression. Several key biological processes in the hippocampus that are needed for neurocognition are affected by dysbiosis caused by acid suppression during stress. Further studies should evaluate associations between microbiota, host gene expression, the abundance of CNS neurocognitive modulators, and their impact on cognition and behavior.

Sodium butyrate alters erythropoietin glycosylation via multiple mechanisms.

Recombinant human erythropoietin (rHuEPO) produced in a human kidney fibrosarcoma cell line, HT1080, was used as a model to study the effects of sodium butyrate (SB) on protein glycosylation. Treatment with 2 mM SB resulted in complex changes with respect to sugar nucleotide pools including an increase in UDP-Gal and a decrease in UDP-GlcNac. In addition, polylactosamine structures present on rHuEPO increased after SB treatment. To determine if these phenotypic changes correlated with changes in mRNA abundance, we profiled mRNA levels over a 24-h period in the presence or absence of SB using oligonucleotide microarrays. By filtering our data through a functional glycomics gene list associated with the processes of glycan degradation, glycan synthesis, and sugar nucleotide synthesis and transport we identified 26 genes with significantly altered mRNA levels. We were able to correlate the changes in message in six of these genes with measurable phenotypic changes within our system including: neu1, b3gnt6, siat4b, b3gnt1, slc17a5, and galt. Interestingly, for the two genes: cmas and gale, our measurable phenotypic changes did not correlate with changes in mRNA expression. These data demonstrate both the utility and pit falls of coupling biochemical analysis with high throughput oligonucleotide microarrays to predict how changes in cell culture environments will impact glycoprotein oligosaccharide content.

Quantitative trait loci for sensitivity to acute ethanol and ethanol consummatory behaviors in rats.

Individuals with a low initial response to alcohol (i.e., ethanol) are at greater risk of developing alcohol abuse or dependence later in life. Similar to humans, individual differences in ethanol sensitivity also can be seen in rats, and several laboratories have used these individual differences to generate selectively bred rats that differ in acute ethanol sensitivity. We have worked with two sets of such rats (Inbred High or Low Alcohol Sensitivity strains, IHAS or ILAS, respectively; Inbred Alcohol Tolerant or Non-Tolerant strains, IAT and IANT, respectively) and have confirmed previously mapped quantitative trait loci (QTL) for these acute differences with the use of recombinant congenic lines; however, the relationship between acute sensitivity and ethanol drinking in these rats has yet to be determined. Thus, here we tested the hypothesis that QTLs underlying variation in initial low sensitivity to ethanol also will modulate variation in ethanol drinking behaviors. Separate groups of selectively inbred parent and congenic rats were tested for the loss of righting response (LORR) and also assessed for ethanol consummatory behavior using either operant self-administration or an intermittent-access two-bottle choice procedure. LORR testing confirmed the presence of a LORR duration QTL in all of the congenics; however, the lack of a corresponding difference in blood ethanol concentration at the regaining of the righting response suggests that these QTLs may be mediating a difference in ethanol metabolism rather than in neuronal sensitivity. IHAS/ILAS-derived congenic rats did not differ from parent rats at any point during operant self-administration. IAT/IANT-derived congenic rats showed small, but significant, increases in ethanol consumption relative to the parent strains only during the initial stages of operant self-administration. In contrast to operant testing, IHAS/ILAS-derived congenic rats showed significantly greater ethanol consumption and preference than parent rats during intermittent-access testing. There were not differences, however, between IAT/IANT congenic and parent rats during intermittent access. These data support the hypothesis that there is a genetic relationship between initial ethanol sensitivity and ethanol consumption, at least for the IHAS/ILAS-derived congenic rats. Our current studies, however, cannot eliminate pharmacokinetic or taste preference factors as contributing to the rats' responses, nor can we eliminate the possibility of a linkage effect because of the fairly large size of the QTL intervals; i.e., distinct genes may be mediating the acute sensitivity and drinking responses.

An Integrated Systems Genetics and Omics Toolkit to Probe Gene Function.

Identifying genetic and environmental factors that impact complex traits and common diseases is a high biomedical priority. Here, we developed, validated, and implemented a series of multi-layered systems approaches, including (expression-based) phenome-wide association, transcriptome-/proteome-wide association, and (reverse-) mediation analysis, in an open-access web server (systems-genetics.org) to expedite the systems dissection of gene function. We applied these approaches to multi-omics datasets from the BXD mouse genetic reference population, and identified and validated associations between genes and clinical and molecular phenotypes, including previously unreported links between Rpl26 and body weight, and Cpt1a and lipid metabolism. Furthermore, through mediation and reverse-mediation analysis we established regulatory relations between genes, such as the co-regulation of BCKDHA and BCKDHB protein levels, and identified targets of transcription factors E2F6, ZFP277, and ZKSCAN1. Our multifaceted toolkit enabled the identification of gene-gene and gene-phenotype links that are robust and that translate well across populations and species, and can be universally applied to any populations with multi-omics datasets.

Short-term selection for acute ethanol tolerance and sensitization from an F2 population derived from the high and low alcohol-sensitive selectively bred rat lines.

Previous studies have identified quantitative trait loci (QTL) in the inbred high and low alcohol-sensitive rat (IHAS1 and ILAS1) strains. The original development of the strains involved selection for ethanol sensitivity based on duration of the loss of the righting reflex (LORR) after a standard dose of ethanol. This paper confirms some of these QTL using a short-term selection procedure based on the difference between the blood ethanol level at LORR and regain of the righting response. An F(2) population of rats was developed by a reciprocal cross of IHAS1 and ILAS1 rats. Selection for five generations was carried out using delta-blood ethanol concentration (dBEC) as the selection trait, where dBEC=BECLR (BEC at loss of righting reflex)-BECRR (BEC at regain of righting reflex). The lines were labeled tolerant (TOL) or sensitive (SENS). Approximately one-third of the offspring for each generation in each line were genotyped using DNA markers that had been previously found to be linked to QTL on chromosomes 1, 2, 5, 12, and 13. By the fifth generation of selection, the lines showed a very large difference in dBEC, BECRR, and duration of LORR; BECLR showed little segregation during the selection, and latency to lose the righting reflex showed none. IHAS allele frequency increased in the SENS line for markers on chromosomes 1, 5, 12, and 13 while ILAS allele frequency increased in the TOL line. These results were in good agreement with the two previous QTL studies. On chromosome 2, the selection resulted in an accumulation of ILAS alleles in both lines. This study provides independent confirmation of the location of QTL on chromosomes 1, 5, 12, and 13 for ethanol sensitivity. It also suggests that genetic differences in duration of LORR are mediated primarily by the dBEC phenotype.

Modulation of GABAA receptors in cerebellar granule neurons by ethanol: a review of genetic and electrophysiological studies.

Cerebellar granule neurons (CGNs) receive inhibitory input from Golgi cells in the form of phasic and tonic currents that are mediated by postsynaptic and extrasynaptic gamma-aminobutyric acid type A (GABAA) receptors, respectively. Extrasynaptic receptors are thought to contain alpha6betaxdelta subunits. Here, we review studies on ethanol (EtOH) modulation of these receptors, which have yielded contradictory results. Although studies with recombinant receptors expressed in Xenopus oocytes indicate that alpha6beta3delta receptors are potently enhanced by acute exposure to low (>or=3 mM) EtOH concentrations, this effect was not observed when these receptors were expressed in Chinese hamster ovary cells. Slice recordings of CGNs have consistently shown that EtOH increases the frequency of phasic spontaneous inhibitory postsynaptic currents (sIPSCs), as well as the tonic current amplitude and noise. However, there is a lack of consensus as to whether EtOH directly acts on extrasynaptic receptors or modulates them indirectly; that is, via an increase in spillover of synaptically released GABA. It was recently demonstrated that an R to Q mutation of amino acid 100 of the alpha6 subunit increases the effect of EtOH on both sIPSCs and tonic current. These electrophysiological findings have not been reproducible in our hands. Moreover, it was shown the alpha6-R100Q mutation enhances sensitivity to the motor-impairing effects of EtOH in outbred Sprague-Dawley rats, but this was not observed in a line of rats selectively bred for high sensitivity to EtOH-induced motor alterations (Alcohol Non-Tolerant rats). We conclude that currently there is insufficient evidence conclusively supporting a direct potentiation of extrasynaptic GABAA receptors following acute EtOH exposure in CGNs.

Confirmation of quantitative trait loci for ethanol sensitivity and neurotensin receptor density in crosses derived from the inbred high and low alcohol sensitive selectively bred rat lines.

RATIONALE: Genetically influenced alcohol sensitivity is thought to be an important risk factor for the development of alcoholism. An effective first step for identifying genes that mediate variation in alcohol sensitivity is through quantitative trait loci (QTL) mapping in model organisms. OBJECTIVE: Fourteen provisional QTLs related to alcohol sensitivity were previously mapped in an F2 derived from the IHAS1 and ILAS1 rat lines. The objective of the current study was to confirm those QTLs in an independently derived F2 and in congenics that were bred for two of the loci. MATERIALS AND METHODS: IHAS1 X ILAS1 F2 (n=450) were tested for alcohol-induced loss of righting reflex (LORR), blood ethanol concentration at regain of righting reflex (BECRR), sensitivity and acute tolerance on the Rotarod, and neurotensin receptor density (NTR1). Rats were genotyped at the 14 candidate loci and QTL mapping was conducted. Reciprocal congenic strains were bred for loci on chromosomes 2 and 5 and tested for LORR and BECRR. RESULTS: Four LORR QTLs were mapped at the suggestive or significant level (chromosomes 2, 5, 12, and 13). BECRR was mapped to chromosomes 5, 12, and 13 either in the original or current experiment. Results of the congenic experiment also support QTLs for LORR and BECRR on chromosomes 2 and 5. QTLs for NTR1 density and behavior on the Rotarod were not confirmed. CONCLUSIONS: QTL mapping in crosses derived from the IHAS1 and ILAS1 has successfully identified loci related to alcohol sensitivity. Recombinant congenics are now being bred to more finely map the confirmed QTLs.

Rapid ethanol tolerance mediated by adaptations in acute tolerance in inbred mouse strains.

It has been postulated that decreased acute sensitivity to ethanol is an important genetically-mediated risk factor for the development of alcoholism. Previous work in mice and rats has indicated that ethanol sensitivity can be reduced in a genotype-dependent manner by a single dose of ethanol 24 h prior to testing, so-called 'rapid' tolerance. The current studies were undertaken to determine if the observed rapid tolerance was mediated by alterations in initial sensitivity or acute functional tolerance (AFT), the two primary components of acute sensitivity. Separate groups of C57BL/6, DBA/2, ILS, and ISS inbred mouse strains were administered a single pretreatment dose of saline or ethanol (5 g/kg). The original and modified versions of the loss of righting reflex test, ethanol-induced hypothermia, and ataxia on a stationary dowel rod were tested 24 h later. Dependent on the test and strain, varying degrees of rapid tolerance were observed; a pronounced sensitization was detected in one case. There was a concomitant increase in the rate and/or magnitude of AFT with little change in initial sensitivity suggesting that rapid tolerance was mediated primarily by alterations in AFT. This conclusion may have implications for the contribution of acute sensitivity to human alcoholism.

Microarray analysis identifies cerebellar genes sensitive to chronic ethanol treatment in PKCgamma mice.

Neuroadaptive changes that occur in the development of ethanol tolerance may be the result of alterations in gene expression. We have shown that PKCgamma wild-type mice develop tolerance to the sedative-hypnotic effects of ethanol after chronic ethanol treatment; whereas, mutant mice do not, making these genotypes a suitable model for identifying changes in gene expression related to tolerance development. Using a two-stage process, several genes were initially identified using microarray analyses of cerebellar tissue from ethanol-treated PKCgamma mutant and wild-type mice. Subsequent confirmation of a subset of these genes using quantitative real time reverse transcriptase polymerase chain reactions (qRT-PCR) was done to verify gene expression changes. A total of 109 genes from different functional classifications were identified in these groups on the microarrays. Eight genes were selected for verification as follows: three, Twik-1, Plp, and Adk2, were chosen as genes related to tolerance; another three, Hsp70.2, Bdnf, and Th, were chosen as genes related to resistance to tolerance; and two genes, JunB and Nur77, were selected as candidate genes sensitive to chronic ethanol. The results from the verification experiments indicated that Twik-1, which codes for a potassium channel, was associated with tolerance and appeared to be dependent on the presence of PKCgamma. No genes were confirmed to be related to resistance to tolerance; however, expression of two of these, Hsp70.2 and Th, were found to be sensitive to chronic ethanol and were added to the transcription factors, JunB and Nur77, confirmed by qRT-PCR, as a subset of genes that respond to chronic ethanol.