Effects of acetate on cerebral blood flow, systemic inflammation, and behavior in alcohol use disorder.

BACKGROUND: Alcohol use disorders (AUDs) are associated with altered regulation of physiological processes in the brain. Acetate, a metabolite of ethanol, has been implicated in several processes that are disrupted in AUDs including transcriptional regulation, metabolism, inflammation, and neurotransmission. To further understand the effects of acetate on brain function in AUDs, we investigated the effects of acetate on cerebral blood flow (CBF), systemic inflammatory cytokines, and behavior in AUD. METHODS: Sixteen participants with AUD were recruited from a nonmedical, clinically managed detoxification center. Each participant received acetate and placebo in a randomly assigned order of infusion and underwent 3T MR scanning using quantitative pseudo-continuous arterial spin labeling. Participants and the study team were blinded to the infusion. CBF values (ml/100 g/min) extracted from thalamus were compared between placebo and acetate using a mixed effect linear regression model accounting for infusion order. Voxel-wise CBF comparisons were set at threshold of p < 0.05 cluster-corrected for multiple comparisons, voxel-level p < 0.0001. Plasma cytokine levels and behavior were also assessed between infusions. RESULTS: Fifteen men and 1 woman were enrolled with Alcohol Use Disorders Identification Test (AUDIT) scores between 13 and 38 with a mean of 28.3 ± 9.1. Compared to placebo, acetate administration increased CBF in the thalamus bilaterally (Left: 51.2 vs. 68.8, p < 0.001; Right: 53.7 vs. 69.6, p = 0.001), as well as the cerebellum, brainstem, and cortex. Older age and higher AUDIT scores were associated with increases in acetate-induced thalamic blood flow. Cytokine levels and behavioral measures did not differ between placebo and acetate infusions. CONCLUSIONS: This pilot study in AUD suggests that during the first week of abstinence from alcohol, the brain's response to acetate differs by brain region and this response may be associated with the severity of alcohol dependence.

Effects of Alcohol and Acetate on Cerebral Blood Flow: A Pilot Study.

BACKGROUND: Acute alcohol produces effects on cerebral metabolism and blood flow. Alcohol is converted to acetate, which serves as a source of energy for the brain and is an agonist at G protein-coupled receptors distributed in different cell types in the body including neurons. Acetate has been hypothesized to play a role in the cerebral blood flow (CBF) response after alcohol ingestion. We tested whether administration of acetate would alter CBF in a pattern similar to or different from that of alcohol ingestion in healthy individuals. METHODS: Twenty-four healthy participants were assigned by convenience to receive either 0.6 g/kg alcohol orally (n = 12) or acetate intravenously (n = 12). For each participant, CBF maps were acquired using an arterial spin labeling sequence on a 3T magnetic resonance scanner after placebo and after drug administration. Whole-brain CBF maps were compared between placebo and drug using a paired t-test, and set at a threshold of p < 0.05 corrected for multiple comparisons (k ≥ 142 voxels, ≥3.78 cm3 ), voxel-level p < 0.005. Intoxication was measured after placebo and drug administration with a Subjective High Assessment Scale (SHAS-7). RESULTS: Compared to placebo, alcohol and acetate were associated with increased CBF in the medial thalamus. Alcohol, but not acetate, was associated with increased CBF in the right orbitofrontal, medial prefrontal and cingulate cortex, and hippocampus. Plasma acetate levels increased following administration of alcohol and acetate and did not differ between the 2 arms. Alcohol, but not acetate, was associated with an increase in SHAS-7 scores (p < 0.001). CONCLUSIONS: Increased thalamic CBF associated with either alcohol or acetate administration suggests that the thalamic CBF response after alcohol could be mediated by acetate. Compared to other brain regions, thalamus may differ in its ability to metabolize acetate or expression of receptors responsive to acetate. Increased prefrontal and limbic CBF associated with alcohol may be linked to alcohol's behavioral effects.

Predictive modeling of miRNA-mediated predisposition to alcohol-related phenotypes in mouse.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that bind messenger RNAs and promote their degradation or repress their translation. There is increasing evidence of miRNAs playing an important role in alcohol related disorders. However, the role of miRNAs as mediators of the genetic effect on alcohol phenotypes is not fully understood. We conducted a high-throughput sequencing study to measure miRNA expression levels in alcohol naïve animals in the LXS panel of recombinant inbred (RI) mouse strains. We then combined the sequencing data with genotype data, microarry gene expression data, and data on alcohol-related behavioral phenotypes such as 'Drinking in the dark', 'Sleep time', and 'Low dose activation' from the same RI panel. SNP-miRNA-gene triplets with strong association within the triplet that were also associated with one of the 4 alcohol phenotypes were selected and a Bayesian network analysis was used to aggregate results into a directed network model. RESULTS: We found several triplets with strong association within the triplet that were also associated with one of the alcohol phenotypes. The Bayesian network analysis found two networks where a miRNA mediates the genetic effect on the alcohol phenotype. The miRNAs were found to influence the expression of protein-coding genes, which in turn influences the quantitative phenotypes. The pathways in which these genes are enriched have been previously associated with alcohol-related traits. CONCLUSION: This work enhances association studies by identifying miRNAs that may be mediating the association between genetic markers (SNPs) and the alcohol phenotypes. It suggests a mechanism of how genetic variants are affecting traits of interest through the modification of miRNA expression.

Voluntary exposure to a toxin: the genetic influence on ethanol consumption.

Ethyl alcohol is a toxin that, when consumed at high levels, produces organ damage and death. One way to prevent or ameliorate this damage in humans is to reduce the exposure of organs to alcohol by reducing alcohol ingestion. Both the propensity to consume large volumes of alcohol and the susceptibility of human organs to alcohol-induced damage exhibit a strong genetic influence. We have developed an integrative genetic/genomic approach to identify transcriptional networks that predispose complex traits, including propensity for alcohol consumption and propensity for alcohol-induced organ damage. In our approach, the phenotype is assessed in a panel of recombinant inbred (RI) rat strains, and quantitative trait locus (QTL) analysis is performed. Transcriptome data from tissues/organs of naïve RI rat strains are used to identify transcriptional networks using Weighted Gene Coexpression Network Analysis (WGCNA). Correlation of the first principal component of transcriptional coexpression modules with the phenotype across the rat strains, and overlap of QTLs for the phenotype and the QTLs for the coexpression modules (module eigengene QTL) provide the criteria for identification of the functionally related groups of genes that contribute to the phenotype (candidate modules). While we previously identified a brain transcriptional module whose QTL overlapped with a QTL for levels of alcohol consumption in HXB/BXH RI rat strains and 12 selected rat lines, this module did not account for all of the genetic variation in alcohol consumption. Our search for QTL overlap and correlation of coexpression modules with phenotype can, however, be applied to any organ in which the transcriptome has been measured, and this represents a holistic approach in the search for genetic contributors to complex traits. Previous work has implicated liver/brain interactions, particularly involving inflammatory/immune processes, as influencing alcohol consumption levels. We have now analyzed the liver transcriptome of the HXB/BXH RI rat panel in relation to the behavioral trait of alcohol consumption. We used RNA-Seq and microarray data to construct liver transcriptional networks, and identified a liver candidate transcriptional coexpression module that explained 24% of the genetic variance in voluntary alcohol consumption. The transcripts in this module focus attention on liver secretory products that influence inflammatory and immune signaling pathways. We propose that these liver secretory products can interact with brain mechanisms that affect alcohol consumption, and targeting these pathways provides a potential approach to reducing high levels of alcohol intake and also protecting the integrity of the liver and other organs.

Unsupervised, Statistically Based Systems Biology Approach for Unraveling the Genetics of Complex Traits: A Demonstration with Ethanol Metabolism.

BACKGROUND: A statistical pipeline was developed and used for determining candidate genes and candidate gene coexpression networks involved in 2 alcohol (i.e., ethanol [EtOH]) metabolism phenotypes, namely alcohol clearance and acetate area under the curve in a recombinant inbred (RI) (HXB/BXH) rat panel. The approach was also used to provide an indication of how EtOH metabolism can impact the normal function of the identified networks. METHODS: RNA was extracted from alcohol-naïve liver tissue of 30 strains of HXB/BXH RI rats. The reconstructed transcripts were quantitated, and data were used to construct gene coexpression modules and networks. A separate group of rats, comprising the same 30 strains, were injected with EtOH (2 g/kg) for measurement of blood EtOH and acetate levels. These data were used for quantitative trait loci (QTL) analysis of the rate of EtOH disappearance and circulating acetate levels. The analysis pipeline required calculation of the module eigengene values, the correction of these values with EtOH metabolism rates and acetate levels across the rat strains, and the determination of the eigengene QTLs. For a module to be considered a candidate for determining phenotype, the module eigengene values had to have significant correlation with the strain phenotypic values and the module eigengene QTLs had to overlap the phenotypic QTLs. RESULTS: Of the 658 transcript coexpression modules generated from liver RNA sequencing data, a single module satisfied all criteria for being a candidate for determining the alcohol clearance trait. This module contained 2 alcohol dehydrogenase genes, including the gene whose product was previously shown to be responsible for the majority of alcohol elimination in the rat. This module was also the only module identified as a candidate for influencing circulating acetate levels. This module was also linked to the process of generation and utilization of retinoic acid as related to the autonomous immune response. CONCLUSIONS: We propose that our analytical pipeline can successfully identify genetic regions and transcripts which predispose a particular phenotype and our analysis provides functional context for coexpression module components.

Uncovering the liver's role in immunity through RNA co-expression networks.

Gene co-expression analysis has proven to be a powerful tool for ascertaining the organization of gene products into networks that are important for organ function. An organ, such as the liver, engages in a multitude of functions important for the survival of humans, rats, and other animals; these liver functions include energy metabolism, metabolism of xenobiotics, immune system function, and hormonal homeostasis. With the availability of organ-specific transcriptomes, we can now examine the role of RNA transcripts (both protein-coding and non-coding) in these functions. A systems genetic approach for identifying and characterizing liver gene networks within a recombinant inbred panel of rats was used to identify genetically regulated transcriptional networks (modules). For these modules, biological consensus was found between functional enrichment analysis and publicly available phenotypic quantitative trait loci (QTL). In particular, the biological function of two liver modules could be linked to immune response. The eigengene QTLs for these co-expression modules were located at genomic regions coincident with highly significant phenotypic QTLs; these phenotypes were related to rheumatoid arthritis, food preference, and basal corticosterone levels in rats. Our analysis illustrates that genetically and biologically driven RNA-based networks, such as the ones identified as part of this research, provide insight into the genetic influences on organ functions. These networks can pinpoint phenotypes that manifest through the interaction of many organs/tissues and can identify unannotated or under-annotated RNA transcripts that play a role in these phenotypes.

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.

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.

Is the alcohol deprivation effect genetically mediated? Studies with HXB/BXH recombinant inbred rat strains.

BACKGROUND: Two features of alcohol addiction that have been widely studied in animal models are relapse drinking following periods of alcohol abstinence and the escalation of alcohol consumption after chronic continuous or intermittent alcohol exposure. The genetic contribution to these phenotypes has not been systematically investigated. METHODS: HXB/BXH recombinant inbred (RI) rat strains were given access to alcohol sequentially as follows: alcohol (10%) as the only fluid for 1 week; alcohol (10%) and water in a 2-bottle choice paradigm for 7 weeks ("pre-alcohol deprivation effect [ADE] alcohol consumption"); 2 weeks of access to water only (alcohol deprivation); and 2 weeks of reaccess to 10% alcohol and water ("post-ADE alcohol consumption"). The periods of deprivation and reaccess to alcohol were repeated 3 times. The ADE was defined as the amount of alcohol consumed in the first 24 hours after deprivation minus the average daily amount of alcohol consumed in the week prior to deprivation. Heritability of the phenotypes was determined by analysis of variance, and quantitative trait loci (QTLs) were identified. RESULTS: All strains showed increased alcohol consumption, compared to the predeprivation period, in the first 24 hours after each deprivation (ADE). Broad-sense heritability of the ADEs was low (ADE1, 9.1%; ADE2, 26.2%; ADE3, 16.3%). Alcohol consumption levels were relatively stable over weeks 2 to 7. Post-ADE alcohol consumption levels consistently increased in some strains and were decreased or unchanged in others. Heritability of pre- and post-ADE alcohol consumption was high and increased over time (week 2, 38.5%; week 7, 51.1%; week 11, 56.8%; week 15, 63.3%). QTLs for pre- and post-ADE alcohol consumption were similar, but the strength of the QTL association with the phenotype decreased over time. CONCLUSIONS: In the HXB/BXH RI rat strains, genotypic variance does not account for a large proportion of phenotypic variance in the ADE phenotype (low heritability), suggesting a role of environmental factors. In contrast, a large proportion of the variance across the RI strains in pre- and post-ADE alcohol consumption is due to genetically determined variance (high heritability).

Genetic markers of comorbid depression and alcoholism in women.

BACKGROUND: Alcohol dependence (AD) is often accompanied by comorbid depression. Recent clinical evidence supports the benefit of subtype-specific pharmacotherapy in treating the population of alcohol-dependent subjects with comorbid major depressive disorder (MDD). However, in many alcohol-dependent subjects, depression is a reactive response to chronic alcohol use and withdrawal and abates with a period of abstinence. Genetic markers may distinguish alcohol-dependent subjects with MDD not tied chronologically and etiologically to their alcohol consumption. In this work, we investigated the association of adenylyl cyclase genes (ADCY1-9), which are implicated in both AD and mood disorders, with alcoholism and comorbid depression. METHODS: Subjects from Vienna, Austria (n = 323) were genotyped, and single nucleotide polymorphisms (1,152) encompassing the genetic locations of the 9 ADCY genes were examined. The Vienna cohort contained alcohol-dependent subjects differentiated using the Lesch Alcoholism Typology. In this typology, subjects are segregated into 4 types. Type III alcoholism is distinguished by co-occurrence of symptoms of depression and by affecting predominantly females. RESULTS: We identified 4 haplotypes associated with the phenotype of Type III alcoholism in females. One haplotype was in a genomic area in proximity to ADCY2, but actually within a lincRNA gene, 2 haplotypes were within ADCY5, and 1 haplotype was within the coding region of ADCY8. Three of the 4 haplotypes contributed independently to Type III alcoholism and together generated a positive predictive value of 72% and a negative predictive value of 78% for distinguishing women with a Lesch Type III diagnosis versus women designated as Type I or II alcoholics. CONCLUSIONS: Polymorphisms in ADCY8 and ADCY5 and within a lincRNA are associated with an alcohol-dependent phenotype in females, which is distinguished by comorbid signs of depression. Each of these genetic locations can rationally contribute to the polygenic etiology of the alcoholism/depression phenotype, and the use of these genetic markers may aid in choosing appropriate and beneficial treatment strategies.

The biometric measurement of alcohol consumption.

BACKGROUND: Proper ascertainment of the history of alcohol consumption by an individual is an important component of medical diagnosis of disease and influences the implementation of appropriate treatment strategies that include prescription of medication, as well as intervention for the negative physical and social consequences of hazardous/harmful levels of alcohol consumption. Biological (biometric) diagnostic tests that provide information on current and past quantity and frequency of alcohol consumption by an individual, prior to onset of organ damage, continue to be sought. METHODS: Platelet monoamine oxidase B (MAO-B) protein was quantitated in 2 populations of subjects who had histories of different levels of alcohol consumption. Levels were assayed by immunoblotting or by ELISA. The development and evaluation of the new ELISA-based measure of platelet MAO-B protein levels is described. RESULTS: One subject population constituted a nontreatment-seeking, cross-sectional subject sample, and the other population was a longitudinally followed, hospitalized group of subjects. An algorithm combining measures of platelet MAO-B protein with the plasma levels of carbohydrate-deficient transferrin (CDT) and with liver enzymes (aspartate aminotransferase or γ-glutamyltransferase [GGT]) can detect hazardous/harmful alcohol use (HHAU) with the highest sensitivity and specificity in the cross-sectional nontreatment-seeking population. In the treatment-seeking population, low MAO-B protein levels at admission are associated with heavy drinking prior to admission, and these protein levels increase over a period of abstinence from alcohol. CONCLUSIONS: The platelet MAO-B protein measurement is particularly effective for male alcohol consumers. The combined use of MAO-B protein measures together with measures of CDT and GGT does, however, improve the diagnostic utility of both markers for ascertaining HHAU in women. Furthermore, measurement of changes in platelet MAO-B protein levels during treatment for alcohol dependence may help monitor the success of the treatment program.

The role of the type 7 adenylyl cyclase isoform in alcohol use disorder and depression.

The translation of extracellular signals to intracellular responses involves a number of signal transduction molecules. A major component of this signal transducing function is adenylyl cyclase, which produces the intracellular "second messenger," cyclic AMP. What was initially considered as a single enzyme for cyclic AMP generation is now known to be a family of nine membrane-bound enzymes, and one cytosolic enzyme. Each member of the adenylyl cyclase family is distinguished by factors that modulate its catalytic activity, by the cell, tissue, and organ distribution of the family members, and by the physiological/behavioral functions that are subserved by particular family members. This review focuses on the Type 7 adenylyl cyclase (AC7) in terms of its catalytic characteristics and its relationship to alcohol use disorder (AUD, alcoholism), and major depressive disorder (MDD). AC7 may be part of the inherited system predisposing an individual to AUD and/or MDD in a sex-specific manner, or this enzyme may change in its expression or activity in response to the progression of disease or in response to treatment. The areas of brain expressing AC7 are related to responses to stress and evidence is available that CRF1 receptors are coupled to AC7 in the amygdala and pituitary. Interestingly, AC7 is the major form of the cyclase contained in bone marrow-derived cells of the immune system and platelets, and in microglia. AC7 is thus, poised to play an integral role in both peripheral and brain immune function thought to be etiologically involved in both AUD and MDD. Both platelet and lymphocyte adenylyl cyclase activity have been proposed as markers for AUD and MDD, as well as prognostic markers of positive response to medication for MDD. We finish with consideration of paths to medication development that may selectively modulate AC7 activity as treatments for MDD and AUD.

Evaluation and characterization of expression quantitative trait analysis methods in the Hybrid Rat Diversity Panel.

The Hybrid Rat Diversity Panel (HRDP) is a stable and well-characterized set of more than 90 inbred rat strains that can be leveraged for systems genetics approaches to understanding the genetic and genomic variation associated with complex disease. The HRDP exhibits substantial between-strain diversity while retaining substantial within-strain isogenicity, allowing for the precise mapping of genetic variation associated with complex phenotypes and providing statistical power to identify associated variants. In order to robustly identify associated genetic variants, it is important to account for the population structure induced by inbreeding. To this end, we investigate the performance of four plausible approaches towards modeling quantitative traits in the HRDP and quantify their operating characteristics. In particular, we investigate three approaches based on genome-wide mixed model analysis, and one approach based on ordinary least squares linear regression. Towards facilitating study planning and design, we conduct extensive simulations to investigate the power of genetic association analyses in the HRDP, and characterize the impressive attained power. In simulation of eQTL data in the HRDP, we find that a mixed model approach that leverages leave-one-chromosome-out kinship estimation attains the highest power while controlling type I error.

Beyond Genes: Inclusion of Alternative Splicing and Alternative Polyadenylation to Assess the Genetic Architecture of Predisposition to Voluntary Alcohol Consumption in Brain of the HXB/BXH Recombinant Inbred Rat Panel.

Post transcriptional modifications of RNA are powerful mechanisms by which eukaryotes expand their genetic diversity. For instance, researchers estimate that most transcripts in humans undergo alternative splicing and alternative polyadenylation. These splicing events produce distinct RNA molecules, which in turn yield distinct protein isoforms and/or influence RNA stability, translation, nuclear export, and RNA/protein cellular localization. Due to their pervasiveness and impact, we hypothesized that alternative splicing and alternative polyadenylation in brain can contribute to a predisposition for voluntary alcohol consumption. Using the HXB/BXH recombinant inbred rat panel (a subset of the Hybrid Rat Diversity Panel), we generated over one terabyte of brain RNA sequencing data (total RNA) and identified novel splice variants (via StringTie) and alternative polyadenylation sites (via aptardi) to determine the transcriptional landscape in the brains of these animals. After establishing an analysis pipeline to ascertain high quality transcripts, we quantitated transcripts and integrated genotype data to identify candidate transcript coexpression networks and individual candidate transcripts associated with predisposition to voluntary alcohol consumption in the two-bottle choice paradigm. For genes that were previously associated with this trait (e.g., Lrap, Ift81, and P2rx4) (Saba et al., Febs. J., 282, 3556-3578, Saba et al., Genes. Brain. Behav., 20, e12698), we were able to distinguish between transcript variants to provide further information about the specific isoforms related to the trait. We also identified additional candidate transcripts associated with the trait of voluntary alcohol consumption (i.e., isoforms of Mapkapk5, Aldh1a7, and Map3k7). Consistent with our previous work, our results indicate that transcripts and networks related to inflammation and the immune system in brain can be linked to voluntary alcohol consumption. Overall, we have established a pipeline for including the quantitation of alternative splicing and alternative polyadenylation variants in the transcriptome in the analysis of the relationship between the transcriptome and complex traits.

Sex Differences in the Brain Transcriptome Related to Alcohol Effects and Alcohol Use Disorder.

There is compelling evidence that sex and gender have crucial roles in excessive alcohol (ethanol) consumption. Here, we review some of the data from the perspective of brain transcriptional differences between males and females, focusing on rodent animal models. A key emerging transcriptional feature is the role of neuroimmune processes. Microglia are the resident neuroimmune cells in the brain and exhibit substantial functional differences between males and females. Selective breeding for binge ethanol consumption and the impacts of chronic ethanol consumption and withdrawal from chronic ethanol exposure all demonstrate sex-dependent neuroimmune signatures. A focus is on resolving sex-dependent differences in transcriptional responses to ethanol at the neurocircuitry level. Sex-dependent transcriptional differences are found in the extended amygdala and the nucleus accumbens. Telescoping of ethanol consumption is found in some, but not all, studies to be more prevalent in females. Recent transcriptional studies suggest that some sex differences may be due to female-dependent remodeling of the primary cilium. An interesting theme appears to be developing: at least from the animal model perspective, even when males and females are phenotypically similar, they differ significantly at the level of the transcriptome.

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.

A long non-coding RNA (Lrap) modulates brain gene expression and levels of alcohol consumption in rats.

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The "hub gene" of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of "Response to Ethanol" emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non-coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype.

Type 7 adenylyl cyclase-mediated hypothalamic-pituitary-adrenal axis responsiveness: influence of ethanol and sex.

Although ethanol has been considered to be an anxiolytic agent, consumption of ethanol has also been shown to increase plasma adrenocorticotropin and glucocorticoids. The corticotrophin-releasing factor (CRF) receptor 1alpha (CRF-R1) is a G protein-coupled receptor that activates adenylyl cyclase (AC), leading to adrenocorticotropin (and subsequently glucocorticoid) release into the circulation. There are nine members of the membrane-bound AC family, and the type 7 AC (AC7) is most sensitive to ethanol, which enhances the responsiveness of AC7 to G protein-coupled receptor activation. We determined the time course of ethanol's effect on plasma adrenocorticotropin and corticosterone levels in male and female AC7 transgenic (Adcy7(huTG)) mice (in which AC7 is overexpressed in neural tissue) and AC7 heterozygous knockdown [Adcy7(+/-)] mice (in which AC7 is underexpressed in neural tissue), and their respective littermate controls [wild type (WT)]. CRF-R1 mRNA and mRNA and protein for different forms of ACs were measured by using gene expression arrays, quantitative reverse transcription-polymerase chain reaction, and immunoblotting in pituitaries of all animals. Our results demonstrated increased levels of AC7 in pituitary of Adcy7(huTG) mice and decreased levels in pituitary of Adcy7(+/-) mice compared with WT animals. Male and female Adcy7(huTG) mice displayed higher plasma adrenocorticotropin and corticosterone levels than WT and/or Adcy7(+/-) mice after ethanol injection. Female mice displayed higher adrenocorticotropin and corticosterone levels after ethanol injection than males, regardless of genotype. The data provide evidence for an integral role of AC7 in the increase of plasma adrenocorticotropin and corticosterone levels during alcohol intoxication.

Phosphorylation regulates removal of synaptic N-methyl-D-aspartate receptors after withdrawal from chronic ethanol exposure.

Alterations in N-methyl-d-aspartate receptor (NMDAR) protein levels or subcellular localization in brain after chronic ethanol exposure may contribute to withdrawal-associated seizures and neurotoxicity. We have investigated synaptic localization of NMDARs in cultured hippocampal pyramidal neurons after prolonged (7 days) exposure to, and acute withdrawal from, 80 mM ethanol using fluorescence immunocytochemistry techniques. After chronic ethanol exposure, there was a significant increase in the clustering of NR1 and NR2B subunits and their colocalization with the synaptic proteins synaptophysin and postsynaptic density protein 95, respectively. There was also increased expression of NR1 variants containing the C2' cassette after chronic ethanol exposure. The ethanol-induced synaptic clustering and colocalization were rapidly reversed within 4 h after ethanol withdrawal. Surface labeling of NR2B subunits suggested that this rapid reversal involved lateral receptor movement to extrasynaptic sites rather than internalization of receptors. Receptor removal from the synapse during ethanol withdrawal was associated with changes in the phosphorylation state of NR2B Ser1480, controlled by the protein kinase CK2. The redistribution of NMDAR to synapses produced by long-term ethanol exposure, as well as the rapid removal during withdrawal, may not only affect neuronal withdrawal hyperexcitability but also may sensitize the system to subsequent synaptic plasticity.