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[
International Worm Meeting,
2021]
Chronic pain affects up to 30% of the population and can be caused by different factors such as injuries or genetic mutations. Chronic pain can take various forms like migraines, different musculoskeletal conditions (low back pain, fibromyalgia), neuropathic pain disorders triggered by cancer or diabetes, as well as visceral pain disorders. Available drugs are not always effective and could trigger many undesirable side effects. In the recent years, genes responsible for those pathologies have been screened in the population with methods, such as linkage analysis and genome wide association studies. Those approaches have revealed a plethora of new candidates but, for the majority of them, we know nothing about their function in the pain pathway. Because studies on pain in human are very limited, we propose C. elegans as a complementary model organism to study conserved nociception genes. In the present study, we first determined a list of C. elegans orthologs for candidate human pain genes reported in the literature and recovered available mutants (109 mutants). We then screened these mutants for alterations in noxious heat-evoked reversals using as high-throughput, computer-assisted behavior analysis pipeline. Using different heat intensities and comparing naive animals with animals repeatedly stimulated with heat, we obtained a set of measures reflecting baseline sensitivity profiles, and the ability to adapt (desensitize) in response to repeated stimuli. Twenty-two mutants displayed significant alterations and could be clustered in different categories, including fast-adapting mutants, non-adapting mutants, and mutants with exacerbated naive sensitivity. As a whole, our study suggests that C. elegans represent a promising model to tackle the function of recently identified human pain-associated genes and set the bases for additional studies on specific candidates. Topics: neurobiology, behavior Keywords: noxious heat avoidance; sensory plasticity; pain modelling
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Glauser, Dominique, Hu, Zehan, Dengjel, Joern, Stumpe, Michael, Rudgalvyte, Martina, Jordan, Aurore, Kressler, Dieter
[
International Worm Meeting,
2021]
Nociception is a conserved process serving as a self-protection system alerting animals of potential damage and underpinning different forms of pain in humans. Some chronic pain conditions may arise from maladaptive modulation in the nociceptive pathway, including within nociceptors, the primary nociceptive sensory neurons. We use Caenorhabditis elegans as a model due to its ability to detect noxious stimuli, perform avoidance behaviors in the form of stimulus-evoked reversals and adapt to repeated stimuli causing a desensitized, analgesia-like state. The worm ortholog of mammalian CaMKI/IV, CMK-1 (calcium/calmodulin-dependent kinase-1) mediates cellular responses to increased calcium levels and is crucial in nociceptors for this avoidance behavior plasticity. However, the downstream elements of the CMK-1 pathway remain unclear. Here, we performed in vitro CMK-1 kinase assays on both peptide and protein from total worm isolates in order to identify direct kinase target candidates via shotgun phosphoproteomics. For in vivo direct/indirect target determination, we carried out stable isotope labeling by amino acids (SILAC) using high-throughput quantitative phosphoproteomics. We used duplex SILAC, where ''light'' 12-carbon and ''heavy'' 13-carbon amino acids were incorporated into nematode proteins for measuring the amounts of phosphorylated proteins in wild type and
cmk-1 null animals. By combining results obtained from these different studies, we were able to ascertain CMK-1 phosphorylation consensus and develop a list of potential CMK-1 targets. Mutants for these candidates were then tested for heat avoidance behavior to determine changes in naive sensitivity to noxious heat and/or adaptation. We used a computer-assisted high-throughput analysis pipeline to quantify heat-evoked reversals in naive animals and animals exposed to repeated stimuli. While wild type animal sensitivity decreased in response to the repeated heat stimuli, some mutant animals failed to adapt. In conclusion, our study reveals several potential CMK-1 targets that may have an important role in behavioral plasticity.
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[
STAR Protoc,
2021]
Standard laboratory culture of <i>Caenorhabditis elegans</i> utilizes solid growth media with a bacterial food source. However, this culture method limits control of food availability and worm population density, factors that impact many life-history traits. Here, we describe liquid-culture protocols for precisely modulating bacterial food availability and population density, facilitating reliable production of arrested L1 larvae, dauer larvae, dietarily restricted worms, or well-fed worms. Worms can be grown in small quantities for standard assays or in the millions for other applications. For complete details on the use and execution of these protocols, please refer to Hibshman etal. (2016), Webster etal. (2018), and Jordan etal. (2019).
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[
International Worm Meeting,
2017]
Long-term effects of extended starvation during L1 arrest have been shown to persist to the F3 generation, suggesting transgenerational epigenetic inheritance. Though effects on lifespan and gene expression have been reported (Rechavi et al), we have only observed relatively subtle transgenerational effects on L1 starvation survival and heat resistance (Jobson, Jordan et al). Furthermore, we found that a minority of starved worms transmitted these effects, requiring phenotype-based sorting of the starved population to detect transgenerational effects among their descendants. In contrast to L1 arrest, larvae can survive dauer arrest for months and the majority of C. elegans in the wild are found as dauers. Persistent effects of dauer arrest have been reported for adults (Hall et al), but transgenerational effects have not been reported. We reasoned that dauer arrest is likely a more ecologically relevant and robust model for transgenerational effects of starvation. Here, we used a liquid culture system carefully controlling worm density and food availability to cause virtually 100% of N2 larvae to enter dauer arrest. These worms remain dauers for months and become reproductive adults when allowed to recover on plates. By assessing various life-history traits, we found that worms subjected to long-term dauer arrest exhibit apparent fitness costs upon recovery, including maternal effects in the F1 generation. However, L1 starvation survival was increased population-wide in the F3 generation (without phenotypic sorting of ancestors), suggesting potentially adaptive transgenerational effects of long-term dauer arrest. This phenotypic effect depended on the length of time the P0 generation spent in dauer arrest, suggesting that dauer formation alone is not sufficient. Notably, the transgenerational effects occurred in the absence of detectable gene expression changes, as if the presumed epigenetic effect on gene expression is distributed over many genes with a small effect on each. This work suggests that experiencing a stress such as starvation can initially be costly, but future generations may exhibit increased fitness in particular circumstances. References: Hall, S.E. et al., A cellular memory of developmental history generates phenotypic diversity in C. elegans. Current Biology, 2010. 20(2): p. 149-155. Jobson, M.A., Jordan J.M. et al., Transgenerational Effects of Early Life Starvation on Growth, Reproduction, and Stress Resistance in Caenorhabditis elegans. Genetics, 2015. 201(1): p. 201-12. Rechavi, O. et al., Starvation-Induced Transgenerational Inheritance of Small RNAs in C. elegans. Cell, 2014. 158(2): p. 277-287.
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[
International Worm Meeting,
2021]
Animals adjust their phenotype to their environment, which is thought to provide a selective benefit under changing conditions. A famous example of such phenotypic plasticity is the slow down of growth and the delay of aging under dietary restriction (DR). Interestingly, parental DR also affects phenotypes of the F1 generation, for example, DR increases the starvation resistance of progeny (Jordan et al., 2019). Molecularly, DR is associated with a global change in proteome expression, including a reduction in ribosome levels and a proteome-wide slow-down of protein turnover (Dhondt et al., 2016; Visscher et al., 2016). Here, we ask if and how parental diet impacts the proteome of the F1 progeny. We will seek causal relationships between intergenerationally transmitted phenotypes and the inherited proteome. Finally, we aim to determine if the parental control of the proteome provides a selective benefit to their progeny. To address these questions, we have established a tool to precisely quantify ribosomal levels by measuring the luminescence from lysis of HiBit tagged to ribosomal proteins. We plan to globally analyze parental effects on the F1 proteome by proteomics and measure the dynamics of proteome changes, growth rates, and reproduction rates in different nutritional conditions by fluorescence live imaging in microchambers. The project will likely provide important insights about the molecular mechanisms and physiological significance of inter-generational inheritance induced by parental diet.
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[
Worm Breeder's Gazette,
1979]
Mutants on the X-chromosome of the dioecious Panagrellus redivivus have been isolated and mapped. Four uncs and a long have been maintained in both sexes. Paralysed, twitchers and dumpies have been obtained, but males fail to mate. Three of the uncs have reduced mobility or coordination, while the fourth unc contracts when tapped on the head, but will not reverse. The long mutant is approximately 30% longer than wild type, with no decrease in body width, Recently a new phenotype, skinny, more than 100% longer and 25% thinner has been isolated by Yvonne Jordan. The frequency of mutation using standard EMS mutagenesis of L4s is 4. 4 X 10+E-4 mutations per visible locus. The number of essential genes on the P. redivivus X-chromosome is calculated at 320 on the basis of the frequency of lethal mutations corrected for double events. The gamma-irradiation induced mutation rate is 9 X 10+E-7 mutations per locus per rad, using a dose-rate of 4.5 Kr/min. Genetically marked L2s were exposed to doses of gamma radiation varying from 5 to 150 Kr. Using target theory applied to males it was calculated that 180, 130 and 1800 X-linked genes in the total P. redivivus genome are required to complete the second, third and final moult, respectively. The kinetics of the failure of females to develop after gamma-irradiation was more indicitive of chromosome breaks rather than point mutation.
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[
International Worm Meeting,
2015]
Dietary restriction delays development, reduces fecundity, and increases lifespan. Here we provide evidence that maternal nutritional status influences provisioning of progeny. In C. elegans, dietary restriction increases progeny size (Harvey and Orbidans, PLOS ONE 2011). We show that progeny of mothers raised under dietary restriction are also buffered against growth-retarding effects of extended L1 arrest (see abstract of J. Jordan et. al.). This suggests that progeny of mothers who experienced limited food are better able to cope with extended starvation. The response to nutrient availability is plastic such that exposure to dietary restriction in late larvae and young adults but not young larvae influences progeny size. Insulin-like signaling plays an important role in regulating embryo size. Disruption of the insulin-like receptor
daf-2 in the soma results in larger embryos. Furthermore, mutations in
daf-2/InsR and
daf-16/FOXO abolish progeny size plasticity in liquid culture-based dietary restriction, with
daf-16 epistatic to
daf-2. This suggests that insulin-like signaling mediates the effects of dietary restriction on progeny size. We find that
skn-1 and
pha-4, two canonical regulators of lifespan extension associated with dietary restriction, are also essential for embryo size increase in an
eat-2 genetic model of dietary restriction on solid media. These findings reveal that while fecundity is decreased in conditions of limited nutrients, the size and starvation resistance of progeny are increased, suggesting that adults anticipate environmental conditions in progeny provisioning. Furthermore, regulation of progeny size requires some of the same genes and pathways controlling lifespan in response to dietary restriction, implying a common response regulates both aging and progeny provisioning.
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[
International Worm Meeting,
2011]
A neuromechanical model of locomotion in C. elegans was recently proposed by Jordan H. Boyle [1]. One of the main results is that both swimming and crawling can be generated by a single neural circuit, reflexively modulated by the environment. This supports the known experimental results showing that different forms of C. elegans forward locomotion (e.g., swimming and crawling) can be described by a modulation of a single biomechanical gait [2]. The modelling result illustrates the importance and the potential of neuromechanical simulations for the analysis of the worm's behaviour.
In order to continue this work, and to make it usable by a broader audience, we have developed a similar neuromechanical model of the worm using CLONES. CLONES (Closed Loop Neural Simulation) is an open source framework for neuromechanical simulations. CLONES implements a communication interface between a neural simulator, called BRIAN [3], and a physics engine for biomedical applications, called SOFA [4]. BRIAN and SOFA are open-source simulators that are easy to use and provide high performance.
Our implementation of the worm's locomotion reproduces the neural model described in [1]. However, there are two key differences between the original physical model and our implementation. Firstly, Boyle's model considers that the body of the worm has zero mass (a low Reynolds number approximation). In contrast, the SOFA simulator allows us to integrate equations with mass and inertia. Secondly, the original model uses rigid rods of fixed length orthogonal to the body axis (approximating the incompressibility of the body due to high internal pressure). In SOFA rigid rods are modeled as springs of very high stiffness.
The physical system simulated in SOFA is described using a XML syntax. The neural network model interpreted by BRIAN is written in Python, using MATLAB-like syntax. Thus, the model is completely interpreted, and it is possible to visualize/interact with the simulation during runtime. Physical environments containing obstacles or chemical concentration gradients can be defined easily.
References
1. Boyle JH: C. elegans locomotion: an integrated approach. PhD thesis, university of Leeds, 2009
2. Berri S, Boyle JH, Tassieri M, Hope IA and Cohen N, Forward locomotion of the nematode C. elegans is achieved through modulation of a single gait HFSP J 3:186, 2009;
3. Goodman DF, Brette R: Brian: a simulator for spiking neural networks in Python. Front Neuroinform 2:5, 2008
4. Allard J, Cotin S, Faure F, Bensoussan PJ, Poyer F, Duriez C, Delingette H, Grisoni L: SOFA - an Open Source Framework for Medical Simulation. Medicine Meets Virtual Reality (MMVR'15), pp. 13-18, 2007.