UNC-18 modulates ethanol sensitivity in Caenorhabditis elegans

Acute ethanol exposure affects the nervous system as a stimulant at low concentrations and as a depressant at higher concentrations, eventually resulting in motor dysfunction and uncoordination. A recent genetic study of two mouse strains with varying ethanol preference indicated a correlation with a polymorphism (D216N) in the synaptic protein Munc18-1. Munc18-1 functions in exocytosis via a number of discrete interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1. We report that the mutation affects binding to syntaxin but not through either a closed conformation mode of interaction or through binding to the syntaxin N terminus. The D216N mutant instead has a specific impairment in binding the assembled SNARE complex. Furthermore, the mutation broadens the duration of single exocytotic events. Expression of the orthologous mutation (D214N) in the Caenorhabditis elegans UNC-18 null background generated transgenic rescues with phenotypically similar locomotion to worms rescued with the wild-type protein. Strikingly, D214N worms were strongly resistant to both stimulatory and sedative effects of acute ethanol. Analysis of an alternative Munc18-1 mutation (I133V) supported the link between reduced SNARE complex binding and ethanol resistance. We conclude that ethanol acts, at least partially, at the level of vesicle fusion and that its acute effects are ameliorated by point mutations in UNC-18.     

Circadian stress tolerance in adult Caenorhabditis elegans

Circadian rhythms control several behaviors through neural networks, hormones and gene expression. One of these outputs in invertebrates, vertebrates and plants is the stress resistance behavior. In this work, we studied the circadian variation in abiotic stress resistance of adult C. elegans as well as the genetic mechanisms that underlie such behavior. Measuring the stress resistance by tap response behavior we found a rhythm in response to osmotic (NaCl LC(50) = 340 mM) and oxidative (H(2)O(2) LC(50) = 50 mM) shocks, with a minimum at ZT0 (i.e., lights off) and ZT12 (lights on), respectively. In addition, the expression of glutathione peroxidase (C11E4.1) and glycerol-3-phosphate dehydrogenase (gpdh-1) (genes related to the control of stress responses) also showed a circadian fluctuation in basal levels with a peak at night. Moreover, in the mutant osr-1 (AM1 strain), a negative regulator of the gpdh-1 pathway, the osmotic resistance rhythms were masked at 350 mM but reappeared when the strain was treated with a higher NaCl concentration. This work demonstrates for the first time that in the adult nematode, C. elegans stress responses vary daily, and provides evidence of an underlying rhythmic gene expression that governs these behaviors.     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.     

Muscle arm development in Caenorhabditis elegans

In several types of animals, muscle cells use membrane extensions to contact motor axons during development. To better understand the process of membrane extension in muscle cells, we investigated the development of Caenorhabditis elegans muscle arms, which extend to motor axons and form the postsynaptic element of the neuromuscular junction. We found that muscle arm development is a highly regulated process: the number of muscle arms extended by each muscle, the shape of the muscle arms and the path taken by the muscle arms to reach the motor axons are largely stereotypical. We also investigated the role of several cytoskeletal components and regulators during arm development, and found that tropomyosin (LEV-11), the actin depolymerizing activity of ADF/cofilin (UNC-60B) and, surprisingly, myosin heavy chain B (UNC-54) are each required for muscle arm extension. This is the first evidence that UNC-54, which is found in thick filaments of sarcomeres, can also play a role in membrane extension. The muscle arm phenotypes produced when these genes are mutated support a 'two-phase' model that distinguishes passive muscle arm development in embryogenesis from active muscle arm extension during larval development.     

Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice

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.     

Factors influencing the development of tolerance to ethanol

The influence of three parameters on the degree of acquired tolerance to ethanol was studied using goldfish as the animal model. These factors were: a) the rate of ethanol administration; b) the repeated performance of the behavioral test, and c) the prior presence of tolerance to a low dose of ethanol. Tolerance, as indicated by the increase in the ethanol brain levels associated with the loss of righting reflex (overturn test), was observed in fish exposed to 0.2, 0.4, or 0.6% (w/v) ethanol for 1 to 24 hours independently of the rate of ethanol administration. Fish exposed to 0.8% (w/v) ethanol developed tolerance only when this ethanol concentration was reached slowly (circa 3 hours). The increase in functional demand by the repeated testing of the fish and the presence of tolerance to a low dose of ethanol (0.4%) did not affect the degree of the observed tolerance in fish left to equilibrate with a 0.8% ethanol dose. The data support the suggestion that motor functions controlled by the central nervous system can adapt to a high concentration of ethanol if an adequate amount of time is allowed for this compensation to occur or if this concentration is reached slowly. Furthermore, in goldfish the degree of acquired tolerance is directly proportional to the concentration of the ethanol during the time of exposure and independent of the previous experience in alcohol solution.     

Cortisol promotes stress tolerance via DAF-16 in Caenorhabditis elegans

In this study, we studied the effects of cortisol and cortisone on the age-related decrease in locomotion in the nematode Caenorhabditis elegans and on the tolerance to heat stress at 35 °C and to oxidative stress induced by the exposure to 0.1% H₂O₂. Changes in mRNA expression levels of C. elegans genes related to stress tolerance were also analyzed. Cortisol treatment restored nematode movement following heat stress and increased viability under oxidative stress, but also shortened worm lifespan. Cortisone, a cortisol precursor, also restored movement after heat stress. Additionally, cortisol treatment increased mRNA expression of the hsp-12.6 and sod-3 genes. Furthermore, cortisol treatment failed to restore movement of daf-16-deficient mutants after heat stress, whereas cortisone failed to restore the movement of dhs-30-deficient mutants after heat stress. In conclusion, the results suggested that cortisol promoted stress tolerance via DAF-16 but shortened the lifespan, whereas cortisone promoted stress tolerance via DHS-30.     

Post-embryonic development in the ventral cord of Caenorhabditis elegans.

56 nerve cells are added to the ventral cord and associated ganglia of Caenorhabditis elegans at about the time of the first larval moult. These cells are produced by the uniform division of 13 neuroblasts followed by a defined pattern of cell deaths. Comparison with the data in the previous paper suggests that there is a relationship between the ancestry of a cell and its function. The significance of programmed cell death is discussed.     

Genes regulating touch cell development in Caenorhabditis elegans

To identify genes regulating the development of the six touch receptor neurons, we screened the F(2) progeny of mutated animals expressing an integrated mec-2::gfp transgene that is expressed mainly in these touch cells. From 2638 mutated haploid genomes, we obtained 11 mutations representing 11 genes that affected the production, migration, or outgrowth of the touch cells. Eight of these mutations were in known genes, and 2 defined new genes (mig-21 and vab-15). The mig-21 mutation is the first known to affect the asymmetry of the migrations of Q neuroblasts, the cells that give rise to two of the six touch cells. vab-15 is a msh-like homeobox gene that appears to be needed for the proper production of touch cell precursors, since vab-15 animals lacked the four more posterior touch cells. The remaining touch cells (the ALM cells) were present but mispositioned. A similar touch cell phenotype is produced by mutations in lin-32. A more severe phenotype; i.e., animals often lacked ALM cells, was seen in lin-32 vab-15 double mutants, suggesting that these genes acted redundantly in ALM differentiation. In addition to the touch cell abnormalities, vab-15 animals variably exhibit embryonic or larval lethality, cell degenerations, malformation of the posterior body, uncoordinated movement, and defective egg laying.     

Touch receptor development and function in Caenorhabditis elegans

Mutations causing a touch-insensitive phenotype in the nematode Caenorhabditis elegans have been the basis of studies on the specification of neuronal cell fate, inherited neurodegeneration, and the molecular nature of mechanosensory transduction. © 1993 John Wiley & Sons, Inc.     

On the control of germ cell development in Caenorhabditis elegans

After hatching, the germ line progenitor cells in C. elegans begin to divide mitotically; later, some of the germ line cells enter meiosis and differentiate into gametes. In the adult, mitotic germ cells, or stem cells, are found at one end (the distal end) and meiotic cells occupy the rest of the elongate gonad. Removal of two somatic gonadal cells, the distal tip cells, by laser microsurgery has a dramatic effect on germ cell development. In either sex, this operation leads to the arrest of mitosis and the initiation of meiosis in germ cells. The function of the distal tip cell in the intact animal appears to be the inhibition of meiosis (or stimulation of mitosis) in nearby germ cells. During development, this permits growth and, in the adult, it maintains the germ line stem cell population. A change in the position of the distal tip cell in the gonad at an early point in development is correlated with a change in the axial polarity of the germ line tissue. This suggests that the localization of the distal tip cell's inhibitory activity at the distal end of the gonad establishes the axial polarity of the germ line tissue in the intact animal.     

Cyclin E expression during development in Caenorhabditis elegans

Our interest in the coordination of cell cycle control and differentiation has led us to investigate the Caenorhabditis elegans cye-1 gene encoding the G(1) cell cycle regulator cyclin E. We have studied the expression and function of cye-1 by using monoclonal antibodies directed against CYE-1 protein, cye-1::GFP reporter genes, and a cye-1 chromosomal deletion mutation. We show that a ubiquitous embryonic pattern of expression becomes restricted and dynamic during postembryonic development. Promoter analysis reveals a relatively small region of cis-acting sequences that are necessary for the complex pattern of expression of this gene. Our studies demonstrate that two other G(1) cell cycle genes, encoding cyclin D and CDK4/6, have similarly compact promoter requirements. This suggests that a relatively simple mechanism of regulation may underlie the dynamic developmental patterns of expression exhibited by these three G(1) cell cycle genes. Our analysis of a new cye-1 deletion allele confirms and extends previous studies of two point mutations in the gene.     

A two-gene balance regulates Salmonella typhimurium tolerance in the nematode Caenorhabditis elegans

Lysozymes are antimicrobial enzymes that perform a critical role in resisting infection in a wide-range of eukaryotes. However, using the nematode Caenorhabditis elegans as a model host we now demonstrate that deletion of the protist type lysozyme LYS-7 renders animals susceptible to killing by the fatal fungal human pathogen Cryptococcus neoformans, but, remarkably, enhances tolerance to the enteric bacteria Salmonella Typhimurium. This trade-off in immunological susceptibility in C. elegans is further mediated by the reciprocal activity of lys-7 and the tyrosine kinase abl-1. Together this implies a greater complexity in C. elegans innate immune function than previously thought.     

Role of cholesterol in germ-line development of Caenorhabditis elegans

We investigated the effects of cholesterol starvation on Caenorhabditis elegans development at both embryonic and post-embryonic stages by examining brood size, embryonic lethality, growth rate, and worm size. The brood sizes of worms grown without cholesterol were substantially reduced in subsequent generations as compared to the control group with cholesterol: 13, 33, and 39% at the first, the second, and the third generation, respectively. The growth rate was also reduced by 20%-26%. Worms became adults after 120-130 hr incubation at 20 degrees C. Embryonic lethality was detected in the range of 1.6%-2.9% as compared to 0.8% of the control group. The percent development from an embryo to an adult was lowered by an average of 10%. Further analyses of germ line development to understand the reduction of brood size revealed that both germ line proliferation and differentiation were affected, and the most striking effect was seen in oogenesis. Defective oogenesis resulted in endomitotic oocytes (Emo, 22% at F1, 26% at F2, and 30% at F3). Thus, cholesterol appears to be required for all developmental stages of C. elegans.     

Caenorhabditis elegans development requires mitochondrial function in the nervous system

The mitochondrial respiratory chain (MRC) supplies the majority of the energy requirements of most eucaryotic cells. A null mutation in the Caenorhabditis elegans nuo-1 gene encoding a subunit of complex I (NADH-ubiquinone oxidoreductase) is lethal, leading to a developmental arrest at the third larval stage. To identify the tissues that regulate development in response to mitochondrial dysfunction, we restored nuo-1 expression with tissue-specific promoters. Only expression of nuo-1 ubiquitously or in the nervous system supported development to the adult stage. Pharyngeal expression of nuo-1 allowed development to proceed to the fourth larval stage. Expression of nuo-1 in the body muscles or in the germline had no effect. Furthermore, only ubiquitous or nervous system expression of nuo-1 allowed exit from the dauer state. Our results indicate that MRC function in the nervous system is needed to send and receive signals that control larval development and exit from dauer.