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Alcohol intoxication can impact learning and this may contribute to the development of problematic alcohol use. In alcohol (ethanol)-induced state-dependent learning (SDL), information learned while an animal is intoxicated is recalled more effectively when the subject is tested while similarly intoxicated than if tested while not intoxicated. When Caenorhabditis elegans undergoes olfactory learning (OL) while intoxicated, the learning becomes state dependent such that recall of OL is only apparent if the animals are tested while intoxicated. We found that two genes known to be required for signal integration, the secreted peptide HEN-1 and its receptor tyrosine kinase, SCD-2, are required for SDL. Expression of hen-1 in the ASER neuron and scd-2 in the AIA neurons was sufficient for their functions in SDL. Optogenetic activation of ASER in the absence of ethanol during learning could confer ethanol state dependency, indicating that ASER activation is sufficient to signal ethanol intoxication to the OL circuit. To our surprise, ASER activation during testing did not substitute for ethanol intoxication, demonstrating that the effects of ethanol on learning and recall rely on distinct signals. Additionally, intoxication-state information could be added to already established OL, but state-dependent OL did not lose state information when the intoxication signal was removed. Finally, dopamine is required for state-dependent OL, and we found that the activation of ASER cannot bypass this requirement. Our findings provide a window into the modulation of learning by ethanol and suggest that ethanol acts to modify learning using mechanisms distinct from those used during memory access.

Multipotent stem and progenitor cells have the capacity to generate a limited array of related cell types. The Caenorhabditis elegans somatic gonadal precursors are multipotent progenitors that generate all 143 cells of the somatic gonad, including complex tissues and specialized signaling cells. To screen for candidate regulators of cell fate and multipotency, we identified transcription factor genes with higher expression in somatic gonadal precursors than in their differentiated sister, the head mesodermal cell. We used RNA interference or genetic mutants to reduce the function of 183 of these genes and examined the worms for defects in the somatic gonadal precursor cell fate or the ability to generate gonadal tissue types. We identify 8 genes that regulate somatic gonadal precursor fate, including the SWI/SNF chromatin remodeling complex gene swsn-3 and the Ci/GLI homolog tra-1, which is the terminal regulator of sex determination. Four genes are necessary for somatic gonadal precursors to generate the correct number and type of descendant cells. We show that the E2F homolog, efl-3, regulates the cell fate decision between distal tip cells and the sheath/spermathecal precursor. We find that the FACT complex gene hmg-4 is required for the generation of the correct number of somatic gonadal precursor descendants, and we define an earlier role for the nhr-25 nuclear hormone receptor-encoding gene, in addition to its previously described role in regulating the asymmetric division of somatic gonadal precursors. Overall, our data show that genes regulating cell fate are largely different from genes regulating developmental potential, demonstrating that these processes are genetically separable.

Multipotent stem and progenitor cells have the capacity to generate a limited array of related cell types. The two C. elegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all 143 cells of the somatic gonad, including complex tissues and specialized signaling cells. To screen for candidate regulators of cell fate and multipotency, we identified transcription factor genes with higher expression in SGPs than in their differentiated sister, the head mesodermal cell. We used RNA interference or genetic mutants to reduce the function of 183 of these genes and examined the worms at two developmental stages. Eight genes were identified that regulate the SGP fate, including the Ci/GLI homolog tra-1, which is the terminal regulator of sex determination. We show that tra-1 regulates SGP fate independently of its role in sex determination, supporting the idea that tra-1 retains ancestral functions. Four genes were necessary for SGPs to generate the correct number and type of descendant cells. We show that the E2F homolog, efl-3, regulates the cell fate decision between distal tip cells and the sheath/spermathecal precursor. We find that the FACT complex gene hmg-4 is required for the generation of the correct number of SGP descendants, and we define an earlier role for the nhr-25 nuclear hormone receptor-encoding gene, in addition to its previously described role in regulating the asymmetric division of SGPs. Overall, our data show that genes regulating cell fate are largely different from genes regulating developmental potential, demonstrating that these processes are genetically separable.

Individual variation in the physiological response to alcohol is predictive of an individual's likelihood to develop alcohol use disorder (AUD). Evidence from diverse model organisms indicates that the levels of long-chain polyunsaturated omega-3 fatty acids (ω-3 LC-PUFAs) can modulate the behavioral response to ethanol and therefore may impact the propensity to develop AUD. While most ω-3 LC-PUFAs come from diet, humans can produce these fatty acids from shorter chain precursors through a series of enzymatic steps. Natural variation in the genes encoding these enzymes has been shown to affect ω-3 LC-PUFA levels. We hypothesized that variation in these genes could contribute to the susceptibility to develop AUD.We identified nine genes (FADS1, FADS2, FADS3, ELOVL2, GCKR, ELOVL1, ACOX1, APOE, and PPARA) that are required to generate ω-3 LC-PUFAs and/or have been shown or predicted to affect ω-3 LC-PUFA levels. Using both set-based and gene-based analyses we examined their association with AUD and two AUD-related phenotypes, alcohol consumption, and an externalizing phenotype.We found that the set of nine genes is associated with all three phenotypes. When examined individually, GCKR, FADS2, and ACOX1 showed significant association signals with alcohol consumption. GCKR was significantly associated with AUD. ELOVL1 and APOE were associated with externalizing.Taken together with observations that dietary ω-3 LC-PUFAs can affect ethanol-related phenotypes, this work suggests that these fatty acids provide a link between the environmental and genetic influences on the risk of developing AUD.

Background The two Caenorhabditis elegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all somatic tissues of the adult reproductive system. The sister cells of the SGPs are two head mesodermal cells (hmcs); one hmc dies by programmed cell death and the other terminally differentiates. Thus, a single cell division gives rise to one multipotent progenitor and one differentiated cell with identical lineage histories. We compared the transcriptomes of SGPs and hmcs in order to learn the determinants of multipotency and differentiation in this lineage. Results We generated a strain that expressed fluorescent markers specifically in SGPs (ehn-3A::tdTomato) and hmcs (bgal-1::GFP). We dissociated cells from animals after the SGP/hmc cell division, but before the SGPs had further divided, and subjected the dissociated cells to fluorescence-activated cell sorting to collect isolated SGPs and hmcs. We analyzed the transcriptomes of these cells and found that 5912 transcripts were significantly differentially expressed, with at least two-fold change in expression, between the two cell types. The hmc-biased genes were enriched with those that are characteristic of neurons. The SGP-biased genes were enriched with those indicative of cell proliferation and development. We assessed the validity of our differentially expressed genes by examining existing reporters for five of the 10 genes with the most significantly biased expression in SGPs and found that two showed expression in SGPs. For one reporter that did not show expression in SGPs, we generated a GFP knock-in using CRISPR/Cas9. This reporter, in the native genomic context, was expressed in SGPs. Conclusions We found that the transcriptional profiles of SGPs and hmcs are strikingly different. The hmc-biased genes are enriched with those that encode synaptic transmission machinery, which strongly suggests that it has neuron-like signaling properties. In contrast, the SGP-biased genes are enriched with genes that encode factors involved in transcription and translation, as would be expected from a cell preparing to undergo proliferative divisions. Mediators of multipotency are likely to be among the genes differentially expressed in SGPs. Electronic supplementary material The online version of this article (10.1186/s12864-019-5821-z) contains supplementary material, which is available to authorized users.

Ethanol-induced transcriptional changes underlie important physiological responses to ethanol that are likely to contribute to the addictive properties of the drug. We examined the transcriptional responses of Caenorhabditis elegans across a timecourse of ethanol exposure, between 30 min and 8 h, to determine what genes and genetic pathways are regulated in response to ethanol in this model. We found that short exposures to ethanol (up to 2 h) induced expression of metabolic enzymes involved in metabolizing ethanol and retinol, while longer exposure (8 h) had much more profound effects on the transcriptome. Several genes that are known to be involved in the physiological response to ethanol, including direct ethanol targets, were regulated at 8 h of exposure. This longer exposure to ethanol also resulted in the regulation of genes involved in cilia function, which is consistent with an important role for the effects of ethanol on cilia in the deleterious effects of chronic ethanol consumption in humans. Finally, we found that food deprivation for an 8-h period induced gene expression changes that were somewhat ameliorated by the presence of ethanol, supporting previous observations that worms can use ethanol as a calorie source.

Background SWI/SNF chromatin remodeling genes are required for normal acute responses to alcohol in C. elegans and are associated with alcohol use disorder in two human populations. In an effort to discover the downstream genes that are mediating this effect, we identified SWI/SNF-regulated genes in C. elegans. Results To identify SWI/SNF-regulated genes in adults, we compared mRNA expression in wild type and swsn-1(os22ts) worms under conditions that produce inactive swsn-1 in mature cells. To identify SWI/SNF-regulated genes in neurons, we compared gene expression in swsn-9(ok1354) null mutant worms that harbor a neuronal rescue or a control construct. RNA sequencing was performed to an average depth of 25 million reads per sample using 50-base, paired-end reads. We found that 6813 transcripts were significantly differentially expressed between swsn-1(os22ts) mutants and wild-type worms and 2412 transcripts were significantly differentially expressed between swsn-9(ok1354) mutants and swsn-9(ok1354) mutants with neuronal rescue. We examined the intersection between these two datasets and identified 603 genes that were differentially expressed in the same direction in both comparisons; we defined these as SWI/SNF-regulated genes in neurons and in adults. Among the differentially expressed genes was cbp-1, a C. elegans homolog of the mammalian CBP/p300 family of histone acetyltransferases. CBP has been implicated in the epigenetic regulation in response to alcohol in animal models and a polymorphism in the human CBP gene, CREBBP, has been associated with alcohol-related phenotypes. We found that cbp-1 is required for the development of acute functional tolerance to alcohol in C. elegans. Conclusions We identified 603 transcripts that were regulated by two different SWI/SNF complex subunits in adults and in neurons. The SWI/SNF-regulated genes were highly enriched for genes involved in membrane rafts, suggesting an important role for this membrane microdomain in the acute alcohol response. Among the differentially expressed genes was cbp-1; CBP-1 homologs have been implicated in alcohol responses across phyla and we found that C. elegans cbp-1 was required for the acute alcohol response in worms.

Author(s): Edwards, Alexis C; Heron, Jon; Hibbeln, Joseph; Schuckit, Marc A; Webb, Bradley T; Hickman, Matthew; Davies, Andrew G; Bettinger, Jill C | Abstract: BackgroundThe levels of the ω-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been associated with alcohol sensitivity in vertebrate and invertebrate model systems, but prior studies have not examined this association in human samples despite evidence of associations between ω-3 LC-PUFA levels and alcohol-related phenotypes. Both alcohol sensitivity and ω-3 LC-PUFA levels are impacted by genetic factors, and these influences may contribute to observed associations between phenotypes. Given the potential for using EPA and DHA supplementation in adjuvant care for alcohol misuse and other outcomes, it is important to clarify how ω-3 LC-PUFA levels relate to alcohol sensitivity.MethodsAnalyses were conducted using data from the Avon Longitudinal Study of Parents and Children. Plasma ω-3 LC-PUFA levels were measured at ages 15.5 and 17.5. Participants reported on their initial alcohol sensitivity using the early drinking Self-Rating of the Effects of Alcohol (SRE-5) scale, for which more drinks needed for effects indicates lower levels of response per drink, at ages 15.5, 16.5, and 17.5. Polygenic liability for alcohol consumption, alcohol problems, EPA levels, and DHA levels was derived using summary statistics from large, publicly available datasets. Linear regressions were used to examine the cross-sectional and longitudinal associations between ω-3 LC-PUFA levels and SRE scores.ResultsAge 15.5 ω-3 LC-PUFA levels were negatively associated with contemporaneous SRE scores and with age 17.5 SRE scores. One modest association (p = 0.02) between polygenic liability and SRE scores was observed, between alcohol problems-based polygenic risk scores (PRS) and age 16.5 SRE scores. Tests of moderation by genetic liability were not warranted.ConclusionsPlasma ω-3 LC-PUFA levels may be related to initial sensitivity to alcohol during adolescence. These data indicate that diet-related factors have the potential to impact humans' earliest responses to alcohol exposure.

BACKGROUND: Complex interactions between environmental and genetic factors influence the risk for developing alcohol use disorder (AUD) in humans. To date, studies of the impact of environment on AUD risk have primarily focused on psychological characteristics or on the effects of developmental exposure to ethanol. We recently observed that modifying levels of the long-chain ω-3 (LC ω-3) fatty acid, eicosapentaenoic acid (EPA), alters acute physiological responses to ethanol in C. elegans. Because mammals derive ω-3 fatty acids from their diet, here we asked if manipulating dietary levels of LC ω-3 fatty acids can affect ethanol-responsive behaviors in mice. METHODS: We used two well-characterized inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), that differ in their responses to ethanol. Age-matched young adult male mice were maintained on isocaloric diets that differed only by being enriched or depleted in LC ω-3 fatty acids. Animals were subsequently tested for acute ethanol sensitivity (locomotor activation and sedation), voluntary consumption, and metabolism. Fat deposition was also determined. RESULTS: We found that dietary levels of LC ω-3s altered ethanol sensitivity and consumption in a genotype-specific manner. Both B6 and D2 animals fed high LC ω-3 diets demonstrated lower ethanol-induced locomotor stimulation than those fed low LC ω-3 diets. Ethanol sedation and ethanol metabolism were greater in D2, but not B6 mice on the high LC ω-3 diet. Conversely, LC ω-3 dietary manipulation altered ethanol consumption in B6, but not in D2 mice. B6 mice on a high LC ω-3 diet consumed more ethanol in a 2-bottle choice intermittent access model than B6 mice on a low LC ω-3 diet. CONCLUSIONS: Because ethanol sensitivity is predictive of risk for developing AUD in humans, our data indicate that dietary LC ω-3 levels should be evaluated for their impact on AUD risk in humans. Further, these studies indicate that genetic background can interact with fatty acids in the diet to significantly alter ethanol-responsive behaviors.

Background Testing for direct gene/SNP replication of association across studies may not capture the true importance of a candidate locus; rather, we suggest that relevant replication across studies may be found at the level of a biological process. We previously observed that variation in two members of the SWI/SNF chromatin remodeling complex is associated with alcohol dependence (AD) in the Irish Affected Sib Pair Study for Alcohol Dependence. Here, we tested for association with alcohol-related outcomes using a set of genes functioning in the SWI/SNF complex in two independent samples. Methods We used a set-based analysis to examine the 29 genes of the SWI/SNF complex for evidence of association with (1) AD in the adult Collaborative Study on the Genetics of Alcoholism (COGA) case/control sample and (2) antisocial behavior, hypothesized to be a genetically-related developmental precursor, in a younger population sample (Spit for Science). Results We found evidence for association of the SWI/SNF complex with AD in COGA (p=0.0435) and more general antisocial behavior in Spit for Science (p=0.00026). The genes that contributed most strongly to the signal in COGA were SS18L1, SMARCD1, BRD7, SMARCB1 and BCL11A. In the Spit for Science sample, ACTB, ARID2, BCL11A, BCL11B, BCL7B, BCL7C, DPF2, and DPF3 all contributed strongly to the signal. Conclusions We detected associations between the SWI/SNF complex and AD in an adult population selected from treatment-seeking probands and antisocial behavior in an adolescent population sample. This provides strong support for a role for SWI/SNF in the development of alcohol-related problems. This article is protected by copyright. All rights reserved.

Background Alcohol use disorder is a serious illness marked by uncontrollable drinking and a negative withdrawal state when not using. Alcohol is one of the most commonly used drugs among adolescent populations. Given that adolescence is a unique developmental stage during which alcohol has long-term effects on future drug-taking behavior; it is essential to understand how early exposure to ethanol during adolescence may affect the abuse liability of the drug later in life. Our studies focused on characterizing how exposure to alcohol in adolescence alters later adult alcohol dependence behaviors, by using well-established mouse models of ethanol drinking. We hypothesized that early exposure to ethanol leads to increased ethanol intake in adults and other behavioral phenotypes that may lead to dependence. Methods We investigated the impact of ethanol drinking in early adolescent C57BL/6J mice using a modified Drinking in the Dark (DID) model. Results Our results showed that exposure to ethanol during adolescence enhanced ethanol intake in adulthood in the DID, and the 2-bottle choice drinking paradigms. In contrast, adult exposure of alcohol did not enhance later alcohol intake. We also conducted tests for ethanol behavioral sensitivity such as loss of righting reflex and anxiety-related behaviors to further elucidate the relationship between adolescent ethanol exposure and enhanced ethanol intake in adult mice. Conclusions Overall, our results suggest that adolescence is a critical period of sensitivity and binge drinking that can lead to lasting changes in ethanol intake in adulthood. Further research will be required in order to more fully examine the neurochemical mechanisms underlying the lasting changes in adulthood.

Evidence suggests that there is an association between polymorphisms in the α5 nicotinic acetylcholine receptor (nAChR) subunit and risk of developing alcohol dependence in humans. The α5 nAChR subunit has also recently been shown to modulate some of the acute response to ethanol in mice. The aim of the current study was to further characterize the role of α5-containing (α5*) nAChRs in acute ethanol responsive behaviors, ethanol consumption and ethanol preference in mice. We conducted a battery of tests in male α5 knockout (KO) mice for a range of ethanol-induced behaviors including hypothermia, hypnosis, and anxiolysis. We also investigated the effects of α5* nAChR on ethanol reward using the Conditioned Place Preference (CPP) assay. Further, we tested the effects of gene deletion on drinking behaviors using the voluntary ethanol consumption in a two-bottle choice assay and Drinking in the Dark (DID, with or without stress) paradigm. We found that deletion of the α5 nAChR subunit enhanced ethanol-induced hypothermia, hypnosis, and an anxiolytic-like response in comparison to wild-type controls. The α5 KO mice showed reduced CPP for ethanol, suggesting that the rewarding properties of ethanol are decreased in mutant mice. Interestingly, Chrna5 gene deletion had no effect on basal ethanol drinking behavior, or ethanol metabolism, but did decrease ethanol intake in the DID paradigm following restraint stress. Taken together, we provide new evidence that α5 nAChRs are involved in some but not all of the behavioral effects of ethanol. Our results highlight the importance of nAChRs as a possible target for the treatment of alcohol dependence.

Among all members of the voltage-gated, TM6 ion channel superfamily, the proteins that constitute calcium- and voltage-gated potassium channels of large conductance (BK) and their coding genes are unique for their involvement in ethanol-induced disruption of normal physiology and behavior. Moreover, in vitro studies document that BK activity is modified by ethanol with an EC

Among all members of the voltage-gated, TM6 ion channel superfamily, the proteins that constitute calcium- and voltage-gated potassium channels of large conductance (BK) and their coding genes are unique for their involvement in ethanol-induced disruption of normal physiology and behavior. Moreover, in vitro studies document that BK activity is modified by ethanol with an EC50~23 mM, which is near blood alcohol levels considered legal intoxication in most states of the USA (0.08 g/dL = 17.4 mM). Following a succinct introduction to our current understanding of BK structure and function in central neurons, with a focus on neural circuits that contribute to the neurobiology of alcohol use disorders (AUD), we review the modifications in organ physiology by alcohol exposure via BK and the different molecular elements that determine the ethanol response of BK in alcohol-naive systems, including the role of an ethanol-recognizing site in the BK-forming slo1 protein, modulation of accessory BK subunits, and their coding genes. The participation of these and additional elements in determining the response of a system or an organism to protracted ethanol exposure is consequently analyzed, with insights obtained from invertebrate and vertebrate models. Particular emphasis is put on the role of BK and coding genes in different forms of tolerance to alcohol exposure. We finally discuss genetic results on BK obtained in invertebrate organisms and rodents in light of possible extrapolation to human AUD.

Background The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol (EtOH)-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic–phenotypic marker mini-white(mini-w), a derivative of the endogenous gene white(w). Whether the mini-w transgenic marker or the endogenous w gene influences behavioral responses to acute EtOH exposure in flies has not been systematically investigated. Methods We manipulated mini-w and w expression via (i) transposons marked with mini-w, (ii) RNAi against mini-w and w, and (iii) a null allele of w. We assessed EtOH sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of EtOH) and an assay based on EtOH-induced sedation. Results In eRING assays, EtOH-induced impairment of climbing correlated inversely with expression of the mini-w marker from a series of transposon insertions. Additionally, flies harboring a null allele of w or flies with RNAi-mediated knockdown of mini-w were significantly more sensitive to EtOH in eRING assays than controls expressing endogenous w or the mini-w marker. In contrast, EtOH sensitivity and rapid tolerance measured in the EtOH sedation assay were not affected by decreased expression of mini-w or endogenous w in flies. Conclusions EtOH sensitivity measured in the eRING assay is noticeably influenced by w and mini-w, making eRING problematic for studies on EtOH-related behavior in Drosophila using transgenes marked with mini-w. In contrast, the EtOH sensitivity assay described here is a suitable behavioral paradigm for studies on EtOH sensitivity and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-w.

The abused volatile solvent toluene shares many behavioral effects with classic central nervous system depressants such as ethanol. Similarities between toluene and ethanol have also been demonstrated using in vitro electrophysiology. Together, these studies suggest that toluene and ethanol may be acting, at least in part, via common mechanisms.We used the genetic model, Caenorhabditis elegans, to examine the behavioral effects of toluene in a simple system, and used mutant strains known to have altered responses to other CNS depressants to examine the involvement of those genes in the motor effects induced by toluene.Toluene vapor brings about an altered pattern of locomotion in wild-type worms that is visibly distinct from that generated by ethanol. Mutants of the slo-1, rab-3 and unc-64 genes that are resistant to ethanol or the volatile anesthetic halothane show no resistance to toluene. A mutation in the unc-79 gene results in hypersensitivity to ethanol, halothane and toluene indicating a possible convergence of mechanisms of the three compounds. We screened for, and isolated, two mutations that generate resistance to the locomotor depressing effects of toluene and do not alter sensitivity to ethanol.In C. elegans, ethanol and toluene have distinct behavioral effects and minimal overlap in terms of the genes responsible for these effects. These findings demonstrate that the C. elegans model system provides a unique and sensitive means of delineating both the commonalities as well as the differences in the neurochemical effects of classical CNS depressants and abused volatile inhalants.

Staphylococcus epidermidis is one of the most common causes of infections of prosthetic heart valves (prosthetic valve endocarditis [PVE]) and an increasingly common cause of infections of native heart valves (native valve endocarditis [NVE]). While S. epidermidis typically causes indolent infections of prosthetic devices, including prosthetic valves and intravascular catheters, S. epidermidis NVE is a virulent infection associated with valve destruction and high mortality. In order to see if the differences in the course of infection were due to characteristics of the infecting organisms, we examined 31 S. epidermidis NVE and 65 PVE isolates, as well as 21 isolates from blood cultures (representing bloodstream infections [BSI]) and 28 isolates from nasal specimens or cultures considered to indicate skin carriage. Multilocus sequence typing showed both NVE and PVE isolates to have more unique sequence types (types not shared by the other groups; 74 and 71%, respectively) than either BSI isolates (10%) or skin isolates (42%). Thirty NVE, 16 PVE, and a total of 9 of the nasal, skin, and BSI isolates were tested for virulence in Caenorhabditis elegans . Twenty-one (70%) of the 30 NVE isolates killed at least 50% of the worms by day 5, compared to 1 (6%) of 16 PVE isolates and 1 (11%) of 9 nasal, skin, or BSI isolates. In addition, the C. elegans survival rate as assessed by log rank analyses of Kaplan-Meier survival curves was significantly lower for NVE isolates than for each other group of isolates ( P < 0.0001). There was no correlation between the production of poly-β(1-6)- N -acetylglucosamine exopolysaccharide and virulence in worms. This study is the first analysis suggesting that S. epidermidis isolates from patients with NVE constitute a more virulent subset within this species.

The mechanisms by which ethanol induces changes in behavior are not well understood. Here, we show that Caenorhabditis elegans loss-of-function mutations in the synaptic vesicle-associated RAB-3 protein and its guanosine triphosphate exchange factor AEX-3 confer resistance to the acute locomotor effects of ethanol. Similarly, mice lacking one or both copies of Rab3A are resistant to the ataxic and sedative effects of ethanol, and Rab3A haploinsufficiency increases voluntary ethanol consumption. These data suggest a conserved role of RAB-3-/RAB3A-regulated neurotransmitter release in ethanol-related behaviors.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C. elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C. elegans.

Alcoholism is a serious health concern. It is known that an individual's acute ethanol response has significant impacts on the development of alcohol dependence. This study investigated what effect the SWI/SNF chromatin remodeling complex has on acute ethanol response phenotypes. SWI/SNF influences transcription by modifying nucleosome position, thus altering gene expression. Using the simple genetic model, Caenorhabditis elegans, we tested one member of the SWI/SNF complex, SWSN-9. We tested its role in ethanol responses and examined behavioral effects. It was hypothesized that SWSN-9 would influence the development of acute functional tolerance (AFT). AFT is defined as a decrease in the intoxicating effects of ethanol during a single continued exposure to a drug, and is indicative of neural adaptation to ethanol effects. Locomotion speed assays were conducted, and we observed the development of AFT by observing an increase in speed over continued ethanol exposure. We compared AFT development of the wild...

Background Despite the profound clinical significance and strong heritability of alcohol use disorder (AUD), we do not yet have a comprehensive understanding of the naturally occurring genetic variance within the human genome that drives its development. This lack of understanding is likely to be due in part to the large phenotypic and genetic heterogeneities that underlie human AUD. As a complement to genetic studies in humans, many laboratories are using the invertebrate model organisms (iMOs) Drosophila melanogaster (fruit fly) and Caenorhabditis elegans (nematode worm) to identify genetic mechanisms that influence the effects of alcohol (ethanol) on behavior. While these extremely powerful models have identified many genes that influence the behavioral responses to alcohol, in most cases it has remained unclear whether results from behavioral–genetic studies in iMOs are directly applicable to understanding the genetic basis of human AUD. Methods In this review, we critically evaluate the utility of the fly and worm models for identifying genes that influence AUD in humans. Results Based on results published through early 2015, studies in flies and worms have identified 91 and 50 genes, respectively, that influence 1 or more aspects of behavioral responses to alcohol. Collectively, these fly and worm genes correspond to 293 orthologous genes in humans. Intriguingly, 51 of these 293 human genes have been implicated in AUD by at least 1 study in human populations. Conclusions Our analyses strongly suggest that the Drosophila and C. elegans models have considerable utility for identifying orthologs of genes that influence human AUD.

Alcohol abuse is a widespread and serious problem. Understanding the factors that influence the likelihood of abuse is important for the development of effective therapies. There are both genetic and environmental influences on the development of abuse, but it has been difficult to identify specific liability factors, in part because of both the complex genetic architecture of liability and the influences of environmental stimuli on the expression of that genetic liability. Epigenetic modification of gene expression can underlie both genetic and environmentally sensitive variation in expression, and epigenetic regulation has been implicated in the progression to addiction. Here, we identify a role for the switching defective/sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex in regulating the behavioral response to alcohol in the nematode Caenorhabditis elegans. We found that SWI/SNF components are required in adults for the normal behavioral response to ethanol and that different SWI/SNF complexes regulate different aspects of the acute response to ethanol. We showed that the SWI/SNF subunits SWSN-9 and SWSN-7 are required in neurons and muscle for the development of acute functional tolerance to ethanol. Examination of the members of the SWI/SNF complex for association with a diagnosis of alcohol dependence in a human population identified allelic variation in a member of the SWI/SNF complex, suggesting that variation in the regulation of SWI/SNF targets may influence the propensity to develop abuse disorders. Together, these data strongly implicate the chromatin remodeling associated with SWI/SNF complex members in the behavioral responses to alcohol across phyla.

Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Here we show that allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of Caenorhabditis elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations.

Background Genetic influences on alcohol dependence (AD) have been well established, with the heritability of AD as estimated in behavioral genetic studies ranging from 50-60%. However, robust genetic association signals in human studies of AD have been limited. As part of the VCU Alcohol Research Center, we use multiple model organism systems (C. Elegans, Drosophila, mouse, and rat) and human studies to advance our understanding of the genetic basis of alcohol related outcomes. Molecular responses to ethanol are likely to be shared across species because signaling mechanisms are evolutionarily conserved. As individual genes and gene networks are associated with ethanol response in model organisms, it is critical to understand their association with human alcohol related phenotypes. In this study we tested the effect of several gene sets identified through model organism research with a range of phenotypes hypothesized to be involved in alcohol-related outcomes in human studies. Methods All the sets and genes were tested using one of the two related software depending if the sample contains independent individuals (Plink set based analysis) or the sample has dependent individuals (Gskat). In all human samples we used imputed data if available. Plink uses algorithm based on significant pruned SNPs (r^2=0.5) at the threshold (p=0.05) keeping LD structure and permuting phenotype randomly between individuals. Gskat performs Family based association test for sets via GEE Kernel Machine score test. We used covariates same as in previous GWAS and other analyses for each of the samples. Results We find evidence that there is some evidence of association in human samples in all three considered gene sets and genes separately for some considered phenotypes. Max number of drinks was strongly associated with several genes in human samples. Externalizing phenotype also showed association with several genes across most human samples. Considering several human samples allows us more preciously determine gene structure affecting alcohol related phenotypes. Discussion In these analyses we tested gene sets with known association in mouse and invertebrate models for the association in human samples using alcohol related phenotypes in an effort to characterize similarities between human phenotypes and animal analogues. It is important to take into account the multiple pathways by which genes may exert an effect on alcohol problems in humans. Analogues between human and animal models will help with explaining the biological and functional reasons creating behavioral problems.

Alcohol abuse is a significant public health problem. Understanding the molecular effects of ethanol is important for the identification of at risk individuals, as well as the development of novel pharmacotherapies. The large conductance calcium sensitive potassium (BK) channel has emerged as an important player in the behavioral response to ethanol in genetic studies in several model organisms and in humans. The BK channel, slo-1, was identified in a forward genetics screen as a major ethanol target in C. elegans for the effects of ethanol on locomotion and egg-laying behaviors. Regulation of the expression of the BK channel, slo, in Drosophila underlies the development of rapid tolerance to ethanol and benzyl alcohol sedation. Rodent expression studies of the BK-encoding KCNMA1 gene have identified regulation of mRNA levels in response to ethanol exposure, and knock out studies in mice have demonstrated that the beta subunits of the BK channel, KCNMB1 and KCNMB4, can modulate ethanol sensitivity of the channel in electrophysiological preparations, and can influence drinking behavior. In human genetics studies, both KCNMA1 and the genes encoding beta subunits of the BK channel have been associated with alcohol dependence. This review describes the genetic data for a role for BK channels in mediating behavioral responses to ethanol across these species.

The Caenorhabditis elegans gene lin-29 encodes a zinc-finger transcription factor that is required for hypodermal cell terminal differentiation and proper vulva morphogenesis. Here we demonstrate that lin-29 is also required in males for productive mating. We show that lin-29 males can perform the early mating behaviors including response to hermaphrodite contact and vulva location, but they do not perform the subsequent steps of vulva attachment via spicule insertion and sperm transfer. Consistent with this observation, we found that lin-29 mutant spicules are on average 43% shorter than wild-type spicules while other male mating structures appear unaltered. In lin-29 mutants, spicule development goes awry after the generation of spicule cells, when spicule morphogenesis occurs in wild-type males. We show that LIN-29 accumulates in many cells of the wild-type male tail, including those that form the spicules. We demonstrate, through analysis of genetic mosaics, that the formation of wild-type-length spicules requires lin-29(+) in the AB.p lineage, the lineage that gives rise to the spicules and other male copulatory structures. Our mosaic analysis also reveals a role for lin-29(+) in the P1 lineage, which mainly produces sex muscles, cells of the somatic gonad, and body wall muscles.

Background A variety of species and experimental designs have been used to study genetic influences on alcohol dependence, ethanol response, and related traits. Integration of these heterogeneous data can be used to produce a ranked target gene list for additional investigation. Results In this study, we performed a unique multi-species evidence-based data integration using three microarray experiments in mice or humans that generated an initial alcohol dependence (AD) related genes list, human linkage and association results, and gene sets implicated in C. elegans and Drosophila. We then used permutation and false discovery rate (FDR) analyses on the genome-wide association studies (GWAS) dataset from the Collaborative Study on the Genetics of Alcoholism (COGA) to evaluate the ranking results and weighting matrices. We found one weighting score matrix could increase FDR based q-values for a list of 47 genes with a score greater than 2. Our follow up functional enrichment tests revealed these genes were primarily involved in brain responses to ethanol and neural adaptations occurring with alcoholism. Conclusions These results, along with our experimental validation of specific genes in mice, C. elegans and Drosophila, suggest that a cross-species evidence-based approach is useful to identify candidate genes contributing to alcoholism.

The development of tolerance to a drug at the level of the neuron reflects a homeostatic mechanism by which neurons respond to perturbations of their function by external stimuli. Acute functional tolerance (AFT) to ethanol is a fast compensatory response that develops within a single drug session and normalizes neuronal function despite the continued presence of the drug. We performed a genetic screen to identify genes required for the development of acute functional tolerance to ethanol in the nematode C. elegans. We identified mutations affecting multiple genes in a genetic pathway known to regulate levels of triacylglycerols (TAGs) via the lipase LIPS-7, indicating that there is an important role for TAGs in the development of tolerance. Genetic manipulation of lips-7 expression, up or down, produced opposing effects on ethanol sensitivity and on the rate of development of AFT. Further, decreasing cholesterol levels through environmental manipulation mirrored the effects of decreased TAG levels. Finally, we found that genetic alterations in the levels of the TAG lipase LIPS-7 can modify the phenotype of gain-of-function mutations in the ethanol-inducible ion channel SLO-1, the voltage- and calcium-sensitive BK channel. This study demonstrates that the lipid milieu modulates neuronal responses to ethanol that include initial sensitivity and the development of acute tolerance. These results lend new insight into studies of alcohol dependence, and suggest a model in which TAG levels are important for the development of AFT through alterations of the action of ethanol on membrane proteins.

Identifying genes that influence behavioral responses to alcohol is critical for understanding the molecular basis of alcoholism and ultimately developing therapeutic interventions for the disease. Using an integrated approach that combined the power of the Drosophila, Caenorhabditis elegans and mouse model systems with bioinformatics analyses, we established a novel, conserved role for chloride intracellular channels (CLICs) in alcohol-related behavior. CLIC proteins might have several biochemical functions including intracellular chloride channel activity, modulation of transforming growth factor (TGF)-β signaling, and regulation of ryanodine receptors and A-kinase anchoring proteins. We initially identified vertebrate Clic4 as a candidate ethanol-responsive gene via bioinformatic analysis of data from published microarray studies of mouse and human ethanol-related genes. We confirmed that Clic4 expression was increased by ethanol treatment in mouse prefrontal cortex and also uncovered a correlation between basal expression of Clic4 in prefrontal cortex and the locomotor activating and sedating properties of ethanol across the BXD mouse genetic reference panel. Furthermore, we found that disruption of the sole Clic Drosophila orthologue significantly blunted sensitivity to alcohol in flies, that mutations in two C. elegans Clic orthologues, exc-4 and exl-1, altered behavioral responses to acute ethanol in worms and that viral-mediated overexpression of Clic4 in mouse brain decreased the sedating properties of ethanol. Together, our studies demonstrate key roles for Clic genes in behavioral responses to acute alcohol in Drosophila, C. elegans and mice.

Ethanol (EtOH) is metabolized by a 2-step process in which alcohol dehydrogenase (ADH) oxidizes EtOH to acetaldehyde, which is further oxidized to acetate by aldehyde dehydrogenase (ALDH). Although variation in EtOH metabolism in humans strongly influences the propensity to chronically abuse alcohol, few data exist on the behavioral effects of altered EtOH metabolism. Here, we used the nematode Caenorhabditis elegans to directly examine how changes in EtOH metabolism alter behavioral responses to alcohol during an acute exposure. Additionally, we investigated EtOH solution osmolarity as a potential explanation for contrasting published data on C. elegans EtOH sensitivity.We developed a gas chromatography assay and validated a spectrophotometric method to measure internal EtOH in EtOH-exposed worms. Further, we tested the effects of mutations in ADH and ALDH genes on EtOH tissue accumulation and behavioral sensitivity to the drug. Finally, we tested the effects of EtOH solution osmolarity on behavioral responses and tissue EtOH accumulation.Only a small amount of exogenously applied EtOH accumulated in the tissues of C. elegans and consequently their tissue concentrations were similar to those that intoxicate humans. Independent inactivation of an ADH-encoding gene (sodh-1) or an ALDH-encoding gene (alh-6 or alh-13) increased the EtOH concentration in worms and caused hypersensitivity to the acute sedative effects of EtOH on locomotion. We also found that the sensitivity to the depressive effects of EtOH on locomotion is strongly influenced by the osmolarity of the exogenous EtOH solution.Our results indicate that EtOH metabolism via ADH and ALDH has a statistically discernable but surprisingly minor influence on EtOH sedation and internal EtOH accumulation in worms. In contrast, the osmolarity of the medium in which EtOH is delivered to the animals has a more substantial effect on the observed sensitivity to EtOH.

Ethanol induces similar behavioral responses in mammals and the fruit fly, Drosophila melanogaster. By coupling assays for ethanol-related behavior to the genetic tools available in flies, a number of genes have been identified that influence physiological responses to ethanol. To enhance the utility of the Drosophila model for investigating genes involved in ethanol-related behavior, we explored the value of an assay that measures the sedative effects of ethanol on negative geotaxis, an evoked locomotor response.We established eRING (ethanol Rapid Iterative Negative Geotaxis) as an assay for quantitating the sedative effects of ethanol on negative geotaxis (i.e., startle-induced climbing). We validated the assay by assessing acute sensitivity to ethanol and rapid ethanol tolerance in several different control strains and in flies with mutations known to disrupt these behaviors. We also used eRING in a candidate screen to identify mutants with altered ethanol-related behaviors.Negative geotaxis measured in eRING assays was dose-dependently impaired by ethanol exposure. Flies developed tolerance to the intoxicating effects of ethanol when tested during a second exposure. Ethanol sensitivity and rapid ethanol tolerance varied across 4 control strains, but internal ethanol concentrations were indistinguishable in the 4 strains during a first and second challenge with ethanol. Ethanol sensitivity and rapid ethanol tolerance, respectively, were altered in flies with mutations in amnesiac and hangover, genes known to influence these traits. Additionally, mutations in the beta integrin gene myospheroid and the alpha integrin gene scab increased the initial sensitivity to ethanol and enhanced the development of rapid ethanol tolerance without altering internal ethanol concentrations.The eRING assay is suitable for investigating genetic mechanisms that influence ethanol sensitivity and rapid ethanol tolerance. Ethanol sensitivity and rapid ethanol tolerance depend on the function of alpha and beta integrins in flies.

Alcohol addiction is a widespread societal problem, for which there are few treatments. There are significant genetic and environmental influences on abuse liability, and understanding these factors will be important for the identification of susceptible individuals and the development of effective pharmacotherapies. In humans, the level of response to alcohol is strongly predictive of subsequent alcohol abuse. Level of response is a combination of counteracting responses to alcohol, the level of sensitivity to the drug and the degree to which tolerance develops during the drug exposure, called acute functional tolerance. We use the simple and well-characterized nervous system of Caenorhabditis elegans to model the acute behavioral effects of ethanol to identify genetic and environmental factors that influence level of response to ethanol. Given the strong molecular conservation between the neurobiological machinery of worms and humans, cellular-level effects of ethanol are likely to be conserved. Increasingly, variation in long-chain polyunsaturated fatty acid levels has been implicated in complex neurobiological phenotypes in humans, and we recently found that fatty acid levels modify ethanol responses in worms. Here, we report that 1) eicosapentaenoic acid, an omega-3 polyunsaturated fatty acid, is required for the development of acute functional tolerance, 2) dietary supplementation of eicosapentaenoic acid is sufficient for acute tolerance, and 3) dietary eicosapentaenoic acid can alter the wild-type response to ethanol. These results suggest that genetic variation influencing long-chain polyunsaturated fatty acid levels may be important abuse liability loci, and that dietary polyunsaturated fatty acids may be an important environmental modulator of the behavioral response to ethanol.

Caenorhabditis elegans vulval development culminates during exit from the L4-to-adult molt with the formation of an opening through the adult hypodermis and cuticle that is used for egg laying and mating. Vulva formation requires the heterochronic gene lin-29, which triggers hypodermal cell terminal differentiation during the final molt. lin-29 mutants are unable to lay eggs or mate because no vulval opening forms; instead, a protrusion forms at the site of the vulva. We demonstrate through analysis of genetic mosaics that lin-29 is absolutely required in a small subset of lateral hypodermal seam cells, adjacent to the vulva, for wild-type vulva formation and egg laying. However, lin-29 function is not strictly limited to the lateral hypodermis. First, LIN-29 accumulates in many non-hypodermal cells with known roles in vulva formation or egg laying. Second, animals homozygous for one lin-29 allele, ga94, have the vulval defect and cannot lay eggs, despite having a terminally differentiated adult lateral hypodermis. Finally, vulval morphogenesis and egg laying requires lin-29 activity within the EMS lineage, a lineage that does not generate hypodermal cells.