The mechanisms by which ethanol induces changes in behavior are not well understood. Here we show that C. elegans loss-of-function mutations in the synaptic vesicle-associated RAB-3 protein and its GTP 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.

Background

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.

Methods

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.

Results

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 four control strains, but internal ethanol concentrations were indistinguishable in the four 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 β integrin gene myospheroid and the α integrin gene scab increased the initial sensitivity to ethanol and enhanced the development of rapid ethanol tolerance without altering internal ethanol concentrations.

Conclusions

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 α and β integrins in flies.

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.

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.

Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin null mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

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.