da Silva AF et al. (2022) Neurotoxicology "JM-20 affects GABA neurotransmission in Caenorhabditis elegans."

Arantes LP, Soares MV, Nunez-Figueredo Y, Cordeiro LM, da Silveira TL, Soares FAA, Baptista FBO, Machado ML, Zamberlan DC, da Silva AF, Rodriguez EO

[Neurotoxicology, 2022]

Along with the discovery of new candidate molecules for pharmaceuticals, several studies have emerged showing different mechanisms of action and toxicological aspects. 3-ethoxycarbonyl-2-methyl-4- (2-nitrophenyl)4,11-dihydro-1H-pyrido [2,3-b] [1,5] benzodiazepine (JM-20) is a hybrid molecule. It is derived from 1,5-benzodiazepines and structurally differentiated by the addition of 1,4-dihydropyridine bonded to the benzodiazepine ring. This gives this molecule potential neuroprotective, antioxidant, and anxiolytic activity. As this is a promising multi-target molecule, further studies are necessary to improve the knowledge about its mechanism of action. In our study, we used Caenorhabditis elegans (C. elegans) to investigate the effects of chronic treatment with JM-20. Nematodes from the wild-type strain (N2) were treated chronically at different concentrations of JM-20. Our results show that JM-20 does not cause mortality, but higher concentrations can delay the development of worms after 48h exposure. We assessed basic behaviors in the worm, and our data demonstrate decreased defecation cycle. Our results suggest that JM-20 acts on the C. elegans GABAergic system because GABA neurotransmission is associated with the worm intestine. We also observed increased locomotor activity and decreased egg-laying after JM-20 treatment. When both behaviors were evaluated in mutants with have reduced levels of GABA (unc-25), this effect is no observed, suggesting the GABAergic modulation. Still, the JM-20 exert similar effect of Diazepam in basic behaviors observed. To reinforce neuromodulatory action, computational analysis was performed, and results showed a JM-20 binding on allosteric sites of nematodes GABA receptors. Overall, this work provided a better understanding of the effects of JM-20 in C. elegans as well as showed the effects of this new molecule on the GABAergic system in this animal model.

Harlow E et al. (2006) CSH Protoc "Fixing Caenorhabditis elegans in Bouin's fixative."

Harlow E, Lane D

[CSH Protoc, 2006]

INTRODUCTIONBouin's fixative is a particularly good choice for worms because it penetrates dense tissues well and is extremely good for fixing antigens. Like all strong fixatives, however, it is unsuitable for some antibody-antigen pairs. In such cases, the length of time in the Bouin's fixative can be shortened, or paraformaldehyde fixation can be used instead.

Killeen M et al. (2002) Dev Biol "UNC-5 function requires phosphorylation of cytoplasmic tyrosine 482, but its UNC-40-independent functions also require a region between the ZU-5 and death domains."

Culotti J, Pawson T, Tong J, Steven R, Killeen M, Krizus A, Scott I

[Dev Biol, 2002]

Members of the UNC-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different UNC-5 domains, we sequenced mutations in seven severe and three weak alleles of unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of UNC-5, wild-type and mutant unc-5::HA transgenes were tested for their ability to rescue the unc-5(e53) null mutant. Our data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, UNC-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, UNC-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to UNC-5 function in vivo. Our data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of UNC-5.

Harlow E et al. (2006) CSH Protoc "Fixing Caenorhabditis elegans in Paraformaldehyde."

Harlow E, Lane D

[CSH Protoc, 2006]

INTRODUCTIONAnother common method for fixing worms is to use paraformaldehyde. This method provides a gentler fixation than the Bouin's method, but often requires the use of collagenase. This method is particularly good for examining adult worms.

Shtonda BB et al. (2006) J Exp Biol "Dietary choice behavior in Caenorhabditis elegans."

Avery L, Shtonda BB

[J Exp Biol, 2006]

Animals have evolved diverse behaviors that serve the purpose of finding food in the environment. We investigated the food seeking strategy of the soil bacteria-eating nematode Caenorhabditis elegans. C. elegans bacterial food varies in quality: some species are easy to eat and support worm growth well, while others do not. We show that worms exhibit dietary choice: they hunt for high quality food and leave hard-to-eat bacteria. This food seeking behavior is enhanced in animals that have already experienced good food. When hunting for good food, worms alternate between two modes of locomotion, known as dwelling: movement with frequent stops and reversals; and roaming: straight rapid movement. On good food, roaming is very rare, while on bad food it is common. Using laser ablations and mutant analysis, we show that the AIY neurons serve to extend roaming periods, and are essential for efficient food seeking.

Hartman PS et al. (1995) International C. elegans Meeting "UV RADIATION INHIBITS CELL DIVISION IN THE P1 LINEAGE MORE THAN IN THE AB LINEAGE"

Buckles D, Hartman PS

[International C. elegans Meeting, 1995]

Two-cell embryos were irradiated with germicidal (254nm) radiation and subsequent cell divisions were determined using Nomarski DIC microscopy. A dose of 30 Jm-2 (which gave ca. 10% survival) delayed P1 and P2 divisions by 27%, versus only 8% for the equivalent AB divisions. Similarly, although exposure to 150 Jm-2 abolished cytokinesis in some AB descendants (resulting in occasional binucleated cells), the next two cell cycles were only 22% longer than in unirradiated controls. Conversely, the P1 and P2 cell cycles averaged 95% longer. The delays in the EMSt, E, and MSt cell cycles were similar to those in the AB lineage, suggesting the longer delays were limited to the P4 progenitors. "Cell-cycle checkpoints" have been documented in other organisms. These afford additional time for restitution of DNA damage. Such checkpoints are apparently largely inoperative in the AB lineage (which generates soma exclusively) but at least partially functional in the cells which give rise to the germ line.

Katzen, Abraham et al. (2015) International Worm Meeting "Economic decision-making and neural correlates of value in C. elegans."

Harbaugh, William, Katzen, Abraham, Lockery, Shawn

[International Worm Meeting, 2015]

Investigation of the neuronal basis of economic decisions would be accelerated by establishing decision making paradigms in simple, genetically tractable organisms, such as the nematode Caenorhabditis elegans. For an organism to be a valid model of economic decision making its choice behavior must be sensitive to: (i) the difference between high and low quality goods, and (ii) the relative cost of those options.Previous work has shown that the nematode worm C. elegans quickly learns to feed on those foods (species of bacteria) that promote higher rates of growth and reproduction. Worms spend more time foraging in patches of Good bacteria (high worm growth rate) versus Mediocre bacteria (moderate growth rate) when equally abundant. Until now, however, it has not been possible to simultaneously present two food choices of different quality and cost. To that end, we have developed an electro-microfluidic device in which a semi-restrained worm forages between contiguous yet discrete fluid streams containing good and mediocre quality food. This arrangement constitutes a two-alternative forced-choice task, analogous to those used in behavioral economics. Electrodes inserted into the device monitor muscular impulses associated with individual swallowing events. Relative consumption of Good and Mediocre food is measured by counting the number of swallowing events in the respective fluid streams. The fraction of total swallowing events in Good vs Mediocre food serves as an index of food preference. Importantly, we can alter the effective prices of the two foods by adjusting the concentration of the bacteria, with price being inversely related to concentration.Here we present behavioral data delineating preference for Good vs Mediocre food across a range of relative prices. We find that worms exposed to the two species of bacteria at equal prices prefer Good bacteria, indicating that feeding preferences are normal in the device. Worms respond to price adjustments as predicted by economic theory in that increasing the relative price of a food leads to a decline in its consumption. In addition, we present calcium-imaging data from sensory neurons showing that they respond to transitions between Good and Mediocre foods, and the amplitude of calcium signal scales with relative food preference. These results show that C. elegans forages in an economic manner, and that relative value is represented at the level of the sensory neurons.

Svendsen BA et al. (1988) Worm Breeder's Gazette "effects of UV radiation on embryonic DNA synthesis and cell division (subtitle: tough guy polymerases from the worm?)."

Reddy J, Hartman PS, Svendsen BA

[Worm Breeder's Gazette, 1988]

Synchronous populations of young embryos were exposed to various fluences of 254 radiation and incubated in the presence of tritiated thymidine. At selected times, samples were processed to determine the effects of irradiation on DNA synthesis (measured as the incorporation of tritium into TCA precipitable material) and cell division [measured by counting nuclei a la Gossett and Hecht (1980. J. Histochem. Cytochem. 28, 507-510)]. Despite the fact that over 50% of embryos failed to develop into adults after fluences of 25 Jm-2 or greater, they displayed control (unirradiated) levels of both cell division and replication at fluences of up to 250 Jm-2. This resistance was not due to UV light attenuation, since both parameters were strongly inhibited in two UV radiation hypersensitive mutants (rad-1 and rad-3) by fluences of 10 Jm-2 and greater. Other systems (e.g., bacteria, mammalian cells, yeast) are much more sensitive to the effects of UV radiation on DNA replication. Analysis of the sizes of newly synthesized DNA in irradiated and unirradiated embryos may provide an explanation for this extreme radiation resistance. Synchronous embryos were irradiated, pulse labeled with tritiated thymidine, and chased for various time intervals. After lysis with proteinase K and SDS, samples were subjected to centrifugation through alkaline sucrose gradients. Inclusion of [14C]-labeled T4 and T7 DNA's allowed determination of molecular weights. Fluences of up to 270 Jm-2 failed to reduce the molecular weight of newly synthesized DNA relative to unirradiated controls. This was unexpected, since damage to the parental strand in other organisms suppresses the size of nascent molecules by preventing elongation of DNA polymerase. It can be calculated, based upon the induction frequency of cyclobutane dimers as well as the size of the newly synthesized DNA, that greater than ten cyclobutane dimers were induced in the template opposite each nascent fragment. This leads to the provocative possibility that a DNA polymerase from C. elegans is capable of trans-lesion synthesis through radiation-induced DNA damage. These data are in keeping with the observation, described in the preceding paragraph, that DNA synthesis and cell division during embryogenesis are refractory to super-lethal doses of UV radiation.

Boris Shtonda et al. (2004) West Coast Worm Meeting "Food-seeking behavior in C. elegans is accomplished by switching modes of locomotion and requires AIY interneurons."

Boris Shtonda, Leon Avery

[West Coast Worm Meeting, 2004]

In a simplified laboratory environment, Caenorhabditis elegans is routinely fed with the intestinal symbiotic bacterium Escherichia coli . In the wild, worms are probably exposed to a variety of bacterial food. How they deal with this choice is unknown. Do worms eat the first type of bacteria they encounter or try to search for food of higher quality? We have shown previously that: 1) when given a choice, worms chose the food of higher quality; 2) they evaluate food via feeding; 3) worms will leave a colony of hard-to-eat food, but will stay in a colony of easy-to-eat food; 4) after exposure to good food, worms tend to leave mediocre food more actively. Here, we show that worms discriminate between good and bad food by regulating two modes of locomotion, known as roaming and dwelling (Fujiwara et al, 2002). On a lawn of mediocre food, roaming is enhanced, on good food dwelling is predominant. Mutants deficient in roaming, such as osm-6 , do not roam on bad food and are deficient in food preference. We found that ttx-3 mutants are also incapable of discriminating between good and bad food. Similarly to osm-6 , ttx-3 tends to dwell even on poor quality food. Both mutants are partially rescued by a mutation known to enhance roaming, egl-4 . The food preference defect of ttx-3 ; osm-6 double is worse than that of either of the singles. TTX-3 is a transcription factor required for the differentiation of the AIY interneurons. It is also expressed in three other neuronal types, AIA, ASI and ADL, where its function is unknown. When AIY is ablated with the laser, worms are partially deficient in food preference and periods of roaming on bad food tend to be shorter. The defect of the AIY-null worms is not as severe as of the ttx-3 mutant, suggesting that TTX-3 functions in other neurons as well to regulate this behavior. In summary, worms find good food by increasing the roaming mode of locomotion on poor food and increasing the dwelling mode of locomotion on good food. Therefore, at least one function of roaming is food seeking. TTX-3 and the AIY neurons function to activate roaming in response to hard-to-eat food and are needed for normal food seeking. Fujiwara et al., Neuron (2002) 36:1091-1102.

Zhang WB et al. (2016) Cell Syst "Extended Twilight among Isogenic C.elegans Causes a Disproportionate Scaling between Lifespan and Health."

Zhang WB, Pincus Z, Sinha DB, Pittman WE, Stroustrup N, Hvatum E

[Cell Syst, 2016]

Although many genetic factors and lifestyle interventions are known to affect the mean lifespan of animal populations, the physiological variation displayed by individuals across their lifespans remains largely uncharacterized. Here, we use a custom culture apparatus to continuously monitor five aspects of aging physiology across hundreds of isolated Caenorhabditis elegans individuals kept in a constant environment from hatching until death. Aggregating these measurements into an overall estimate of senescence, we find two chief differences between longer- and shorter-lived individuals. First, though long- and short-lived individuals are physiologically equivalent in early adulthood, longer-lived individuals experience a lower rate of physiological decline throughoutlife. Second, and counter-intuitively, long-lived individuals have a disproportionately extended "twilight" period of low physiological function. Whilelonger-lived individuals experience more overall days of good health, their proportion of good to bad health, and thus their average quality of life, is systematically lower than that of shorter-lived individuals. We conclude that, within a homogeneous population reared under constant conditions, the period of early-life good health is comparatively uniform, and the most plastic period in the aging process is end-of-life senescence.