Many organisms respond to food deprivation by altering their pattern of movement, often in ways that appear to facilitate dispersal. While the behavior of the nematode C. elegans in the presence of attractants has been characterized, long-range movement in the absence of external stimuli has not been examined in this animal. Here we investigate the movement pattern of individual C. elegans over times of ∼1 hour after removal from food, using two custom imaging set-ups that allow us to track animals on large agar surfaces of 22 cm×22 cm. We find that a sizeable fraction of the observed trajectories display directed motion over tens of minutes. Remarkably, this directional persistence is achieved despite a local orientation memory that decays on the scale of about one minute. Furthermore, we find that such trajectories cannot be accounted for by simple random, isotropic models of animal locomotion. This directional behavior requires sensory neurons, but appears to be independent of known sensory signal-transduction pathways. Our results suggest that long-range directional behavior of C. elegans may not be driven by sensory cues.
Ethanol is metabolized by a two-step process in which alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde, which is further oxidized to acetate by aldehyde dehydrogenase (ALDH). Although variation in ethanol metabolism in humans strongly influences the propensity to chronically abuse alcohol, few data exist on the behavioral effects of altered ethanol metabolism. Here, we used the nematode C. elegans to directly examine how changes in ethanol metabolism alter behavioral responses to alcohol during an acute exposure. Additionally, we investigated ethanol solution osmolarity as a potential explanation for contrasting published data on C. elegans ethanol sensitivity.
We developed a gas chromatography assay and validated a spectrophotometric method to measure internal ethanol in ethanol-exposed worms. Further, we tested the effects of mutations in ADH and ALDH genes on ethanol tissue accumulation and behavioral sensitivity to the drug. Finally, we tested the effects of ethanol solution osmolarity on behavioral responses and tissue ethanol accumulation.
Only a small amount of exogenously applied ethanol 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 ethanol concentration in worms and caused hypersensitivity to the acute sedative effects of ethanol on locomotion. We also found that the sensitivity to the depressive effects of ethanol on locomotion is strongly influenced by the osmolarity of the exogenous ethanol solution.
Our results indicate that ethanol metabolism via ADH and ALDH has a statistically discernable but surprisingly minor influence on ethanol sedation and internal ethanol accumulation in worms. In contrast, the osmolarity of the medium in which ethanol is delivered to the animals has a more substantial effect on the observed sensitivity to ethanol.
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, C. 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 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.
alcohol; invertebrate; vertebrate; sensitivity; tolerance; genetics
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, C. 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.
toluene; ethyl benzene; ethanol; halothane; behavior; genetics; C. elegans
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.
RAB-3/RAB3A; AEX-3; presynaptic; neurotransmitter release; ethanol resistance; ethanol consumption
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 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.
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.
behavior; alcohol; genetics
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.
Alcohol has a wide variety of effects on physiology and behavior. One of the most well-recognized behavioral effects is disinhibition, where behaviors that are normally suppressed are displayed following intoxication. A large body of evidence has shown that alcohol-induced disinhibition in humans affects attention, verbal, sexual, and locomotor behaviors. Similar behavioral disinhibition is also seen in many animal models of ethanol response, from invertebrates to mammals and primates. Here we describe several examples of disinhibition in the nematode C. elegans. The nematode displays distinct behavioral states associated with locomotion (crawling on land and swimming in water) that are mediated by dopamine. On land, animals crawl and feed freely, but these behaviors are inhibited in water. We found that additional behaviors, including a variety of escape responses are also inhibited in water. Whereas alcohol non-specifically impaired locomotion, feeding, and escape responses in worms on land, alcohol specifically disinhibited these behaviors in worms immersed in water. Loss of dopamine signaling relieved disinhibition of feeding behavior, while loss of the D1-like dopamine receptor DOP-4 impaired the ethanol-induced disinhibition of crawling. The powerful genetics and simple nervous system of C. elegans may help uncover conserved molecular mechanisms that underlie alcohol-induced disinhibition of behaviors in higher animals.
The regulation of energy balance involves an intricate interplay between neural mechanisms that respond to internal and external cues of energy demand and food availability. Compelling data have implicated the neurotransmitter dopamine as an important part of body weight regulation. However, the precise mechanisms through which dopamine regulates energy homeostasis remain poorly understood. Here, we investigate mechanisms through which dopamine modulates energy storage. We showed that dopamine signaling regulates fat reservoirs in Caenorhabditis elegans. We found that the fat reducing effects of dopamine were dependent on dopaminergic receptors and a set of fat oxidation enzymes. Our findings reveal an ancient role for dopaminergic regulation of fat and suggest that dopamine signaling elicits this outcome through cascades that ultimately mobilize peripheral fat depots.
In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson’s disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying.
Characterizing and understanding the complex spectrum of lipids in higher organisms lags far behind our analysis of genome and transcriptome sequences. Here we generate and evaluate comprehensive lipid profiles (>200 lipids) of 92 inbred lines from five different Drosophila melanogaster populations. We find that the majority of lipid species are highly heritable, and even lipids with odd-chain fatty acids, which cannot be generated by the fly itself, also have high heritabilities. Abundance of the endosymbiont Wolbachia, a potential provider of odd-chained lipids, was positively correlated with this group of lipids. Additionally, we show that despite years of laboratory rearing on the same medium, the lipid profiles of the five geographic populations are sufficiently distinct for population discrimination. Our data predicts a strikingly different membrane fluidity for flies from the Netherlands, which is supported by their increased ethanol tolerance. We find that 18% of lipids show strong concentration differences between males and females. Through an analysis of the correlation structure of the lipid classes, we find modules of co-regulated lipids and begin to associate these with metabolic constraints. Our data provide a foundation for developing associations between variation in lipid composition with variation in other metabolic attributes, with genome-wide variation, and with metrics of health and overall reproductive fitness.
Glutathione (GSH) and GSH-dependent enzymes play a key role in cellular detoxification processes that enable organism to cope with various internal and environmental stressors. However, it is often not clear, which components of the complex GSH-metabolism are required for tolerance towards a certain stressor. To address this question, a small scale RNAi-screen was carried out in Caenorhabditis elegans where GSH-related genes were systematically knocked down and worms were subsequently analysed for their survival rate under sub-lethal concentrations of arsenite and the redox cycler juglone. While the knockdown of γ-glutamylcysteine synthetase led to a diminished survival rate under arsenite stress conditions, GSR-1 (glutathione reductase) was shown to be essential for survival under juglone stress conditions. gsr-1 is the sole GSR encoding gene found in C. elegans. Knockdown of GSR-1 hardly affected total glutathione levels nor reduced glutathione/glutathione disulphide (GSH/GSSG) ratio under normal laboratory conditions. Nevertheless, when GSSG recycling was impaired by gsr-1(RNAi), GSH synthesis was induced, but not vice versa. Moreover, the impact of GSSG recycling was potentiated under oxidative stress conditions, explaining the enormous effect gsr-1(RNAi) knockdown had on juglone tolerance. Accordingly, overexpression of GSR-1 was capable of increasing stress tolerance. Furthermore, expression levels of SKN-1-regulated GSR-1 also affected life span of C. elegans, emphasising the crucial role the GSH redox state plays in both processes.
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.
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.
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.
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.