Trojanowski, Nicholas et al. (2013) International Worm Meeting "Multiple cholinergic pathways for excitation of the Caenorhabditis elegans pharynx."

Trojanowski, Nicholas, Padovan-Merhar, Olivia, Fang-Yen, Christopher, Raizen, David

[International Worm Meeting, 2013]

C. elegans feeds through rhythmic contractions of its pharynx, a neuromuscular pump innervated by a network of 20 neurons. Laser ablation studies have provided key insights into how the pharyngeal nervous system regulates feeding. However, these experiments only permit unidirectional and permanent manipulation of circuit function. To overcome these limitations and study the function of the pharyngeal nervous system, we developed a technique in which individual pharyngeal neurons in immobilized worms are optogenetically manipulated using selective illumination by a laser beam shaped by a digital micromirror device, while machine vision is used to quantify behavioral changes. As expected from ablation data, we found that excitation of the two cholinergic MC neurons causes rapid pumping. Activation of the cholinergic M2 neurons, for which no role has been reported, also causes rapid pumping. This effect is decreased but not abolished when MC is ablated, suggesting that M2 can act independently of MC. Stimulation of cholinergic I1 neurons, which connect the somatic and pharyngeal nervous systems and are anatomically defined to synapse on the MC and M2 neurons, also induces pumping. I1 excitation after ablation of both MC and M2 does not increase pump rate, demonstrating that I1 excites pumping through these two neurons. Previous work indicates that MC stimulates pumping via nicotinic neurotransmission. Surprisingly, in worms lacking the nicotinic receptor subunit EAT-2, excitation of MC or M2 still increases pumping rate. This effect persists in eat-18 mutants, which lack pharyngeal nicotinic neurotransmission, but not unc-17 mutants, which are defective in acetylcholine release, suggesting that MC and M2 can stimulate muscle though a non-nicotinic cholinergic mechanism. MC and M2 can each still excite pumping when the other is ablated in an eat-18 background, suggesting they both act through this cholinergic non-nicotinic pathway. We are identifying other cholinergic receptors involved in feeding and exploring the function of I1 in connecting the pharyngeal and somatic nervous systems.

Trojanowski, Nicholas et al. (2015) International Worm Meeting "Distinct mechanisms underlie two types of Caenorhabditis elegans sleep."

Raizen, David, Trojanowski, Nicholas, Fang-Yen, Christopher, Nelson, Matthew, Flavell, Steven

[International Worm Meeting, 2015]

Electrophysiological recordings have enabled identification of physiologically distinct yet behaviorally similar states of mammalian sleep. In contrast, sleep states in non-mammalian animals have generally been described behaviorally, and therefore non-mammalian sleep is often identified as a homogenous state characterized by quiescence of feeding and locomotion, reduced responsiveness, and rapid reversibility. In C .elegans, sleep has been described under two conditions: developmentally timed sleep (DTS or lethargus) occurs during molting transitions, while stress-induced sleep (SIS) occurs in response to exposure to cellular stress during adulthood. Behaviorally, DTS and SIS appear identical, as during both states feeding and locomotion cease and arousal threshold is increased. We used optogenetic manipulations of neuronal and muscular activity, pharmacology, and genetic perturbations to uncover circuit and molecular mechanisms of DTS and SIS. We find that locomotion quiescence induced by DTS- and SIS-associated neuropeptides (NLP-22 and FLP-13, respectively) occurs via their action on the nervous system. However, in mutants with activated Gs signaling, overexpression of NLP-22 or FLP-13 causes distinct locomotion phenotypes, suggesting that their neuronal target(s) and/or molecular mechanisms differ. Feeding quiescence during DTS results from a loss of pharyngeal muscle excitability, whereas feeding quiescence during SIS results from a loss of excitability in the nervous system. Together these results indicate that, as in mammals, distinct types of sleep in C. elegans are subserved by different mechanisms. .

Yeon, Jihye et al. (2021) MicroPubl Biol

Sengupta, Piali, Takeishi, Asuka, Yeon, Jihye

[MicroPubl Biol, 2021]

Neuronal networks can achieve similar outputs via distinct underlying circuit mechanisms (Beverly et al., 2011; Marder et al., 2015; Saideman et al., 2007; Trojanowski et al., 2014; Wang et al., 2019). This degeneracy allows networks to maintain robustness without compromising functional flexibility (Cropper et al., 2016; Edelman and Gally, 2001). Since the contribution of degenerate neuronal pathways is likely to be revealed under defined genetic or environmental conditions, it is challenging to identify and describe the contributions of such pathways to neuronal circuit function.

Rothstein M et al. (1969) "Culture methods and nutrition of nematodes and Acanthocephala."

Nicholas WL, Rothstein M

[1969]

In order to study properly the nutrition and culture of nematodes, it is desirable to establish the organisms in axenic culture. Only in this way can the metabolic abilities of the nematodes be separated from those of coexisting and interacting organisms. One may settle for a mono-axenic culture, but the best way to attain this is to obtain axenic nematodes and then add the second organism or tissue, for example, alfalfa callus tissue for plant parasitic nematodes (Krusberg, 1961). This chapter will devote itself, in the main, to recent work on the culture and nutrition of nematodes, free-living and parasitic, and will refer only in passing to work already thoroughly reviewed (Dougherty et al., 1959; Nicholas, et al., 1959; Dougherty, 1960).

Chavez-Perez, Carlos et al. (2021) MicroPubl Biol

Rohacek, Alex M, Raizen, David M, Jafari, Niusha, Chavez-Perez, Carlos, Keenan, Brendan T

[MicroPubl Biol, 2021]

Like other animals, the nematode C. elegans exhibits reduced movement and sleep in response to sickness, which can be induced by exposure to high temperatures (Hill et al. 2014; Nelson et al. 2014) ultraviolet light (DeBardeleben et al. 2017), and other stressful exposures (Hill et al. 2014; Goetting et al. 2020). This response has been termed Stress/Sickness-Induced Sleep (SIS) (Hill et al. 2014; Trojanowski and Raizen 2016). Exposure to the stressor leads to quiescence in part via release of the cytokine Epidermal Growth Factor (EGF) (Hill et al. 2014; Konietzka et al. 2020), which is encoded by the gene lin-3 (Hill and Sternberg 1992). EGF activates the ALA and RIS neurons, which then release their respective neuropeptides to effect reduced movement and behavioral quiescence (Konietzka et al. 2020).

Delia M O'Rourke et al. (2005) International Worm Meeting "Analysis of gene expression changes in C. elegans following infection with Microbacterium nematophilum."

Delia M O'Rourke, Maria Demidova, Jonathan Hodgkin, Richard Mott, Dilair Baban

[International Worm Meeting, 2005]

Microbacterium nematophilum is a bacterial pathogen of C. elegans (Hodgkin et al, 2000) that adheres to the rectum and post-anal region of the worm causing swelling of the underlying hypodermal tissue, mild constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype. C. elegans responds to this infection in part through activation of an ERK MAP kinase cascade which mediates tail swelling and prevents severe constipation (Nicholas and Hodgkin, 2004). To identify the downstream transcriptional changes that occur in C. elegans during this host/pathogen interaction we have conducted a genome wide analysis of gene expression using Affymetrix gene chips. We infected a synchronised population of larval C. elegans in liquid culture for 6hrs before harvesting the worms and extracting RNA. Our control sample was an identical experiment using an avirulent M. nematophilum generated in our laboratory by Tanya Akimkina and Steve Curnock. Analysis of data from triplicate microarray experiments has identified a number of statistically significant gene clusters whose expression changes upon infection. Clusters containing up-regulated genes are located on chromosomes IV and V and include C-type lectins, lysozyme-like proteins and proteins containing metridin-like ShK toxin and DUF141 domains. An RNAi feeding screen of these and other induced genes has identified a number that affect the Dar response. The microarray data suggest that although similar functional domains are found in proteins induced by both M. nematophilum and other pathogens, the response of C. elegans to M. nematophilum is specific. Hodgkin, J. Kuwabara, P. E. Corneliussen, B. Current Biology 200010 ;1615-1618 Nicholas, HR, Hodgkin, J. Current Biology 2004 14 ;1256-1261

Partridge, Frederick A. et al. (2009) International Worm Meeting "Investigation of the mechanism of infection induced tail swelling."

Partridge, Frederick A., Hodgkin, Jonathan

[International Worm Meeting, 2009]

When C. elegans and certain other rhabditid nematodes are infected by various bacterial pathogens, notably the coryneform Microbacterium nematophilum, they respond by altering the development of the hindgut, producing a swollen tail. This is a protective innate immune response [1]. Our lab has previously shown that tail swelling involves activation of the ERK MAPK cascade in the rectal epithelial cells. The mechanism of tail swelling has been hard to address in genetic screens as mutants that abolish infectability are much more common than mutants affecting the response to infection. To avoid this problem we have stably expressed activated MAP kinase pathway genes in the hindgut, which phenocopies tail swelling in the absence of infection. This has allowed us to screen for modifiers of tail swelling without the confounding effects of susceptibility to infection. Results of RNAi and forward genetic screens will be presented. [1] Nicholas and Hodgkin (2004) Curr Biol 14 1256-61.

Huang H et al. (2017) Bio Protoc "Measuring Caenorhabditis elegans Sleep During the Transition to Adulthood Using a Microfluidics-based System."

Hart AC, Singh K, Huang H

[Bio Protoc, 2017]

C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski & Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During lethargus, animals cycle between periods of immobility (sleep bouts) and periods of active locomotion (motion bouts). Sleep bouts resemble sleep in other species based on behavioral criteria, including cessation of feeding and locomotion, increased arousal threshold for response to sensory stimulation, rapid reversibility, and homeostatic response to sleep loss. Several assays have been developed to study sleep in C. elegans (Belfer et al., 2013; Bringmann, 2011; Nelson et al., 2013; Raizen et al., 2008). Here, we contribute a detailed protocol for assessment of C. elegans sleep during lethargus, which has been used successfully by many research groups, incorporating simple microfluidic chambers, a low cost camera with lighting system, and computational analysis based on image subtraction. We note that this system could be easily adapted to assess sleep in any small animal.

Statzer, C.A. et al. (2017) International Worm Meeting "Assessing collagen levels during aging and longevity."

Statzer, C.A., Ewald, C.Y.

[International Worm Meeting, 2017]

Collagens are a major component of the extracellular matrix (ECM) and account for 30% of the total human protein mass. As we grow older our collagen mass declines 1% per year in our skin. This decrease is likely due to damage and degradation of collagen as well as transcriptional down-regulation of collagen expression with advanced age. Here, we determine the total collagen content and its decline during aging in C. elegans. We exploit the common post-translation modification of collagen fibers, which contain a hydroxylated form of the amino acid proline. The hydroxylation stabilizes the helical fold of collagens and can be utilized to specifically quantify their abundance. Interestingly, long-lived C. elegans show higher collagen protein levels compared to wild type. We are using publicly available expression profiles to identify collagens that show a progressive decline in mRNA expression levels during aging in wild type, but an upregulation in long-lived animals. We are employing the lifespan machine as developed by Dr. Nicholas Stroustrup to assess the requirements for these candidate collagens for longevity. Taken together, this work aims to elucidate how core ECM components are differentially regulated in normal and long-lived C.elegans and how the ECM itself is modulating the lifespan of an organism.

Coleman-Hulbert, A.L. et al. (2017) International Worm Meeting "Automation of the Caenorhabditis Intervention Testing Program."

Garret, T., Fulger, A.C., Lithgow, G.J., Johnson, E., Kish, J.L., Banse, S., Sedore, C.A., Presley, M.P., Driscoll, M., Harinath, G., Phillips, P.C., Lucanic, M., Falkowski, R., Chen, E., Blue, B.W., Crist, A.B., Hall, D., Coleman-Hulbert, A.L., Plummer, T.W.

[International Worm Meeting, 2017]

The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional consortium supported by the National Institutes of Aging and tasked with identifying robust life-extending compounds using nematodes from the genus Caenorhabditis. CITP partners at Rutgers University, the University of Oregon, and the Buck Institute for Aging Research have streamlined efforts and reproduced findings from 22 diverse strains of Caenorhabditis under ten separate compound interventions. Our work highlights the necessity of replication of longevity analysis, and our intention is to greatly expand the scale of our assays in order to keep up with demand from the aging research community. The C. elegans Lifespan Machine developed by Nicholas Stroustrup (Fontana Lab, Harvard) holds a great deal of promise as a high-throughput longevity approach. The C. elegans Lifespan Machine uses typical office scanners modified to hold petri plates containing nematodes. Through hourly image capture and later image processing, the Lifespan Machine automates the process of gathering lifespan data while providing tight temporal scaling. Here, we compare manual and automated lifespan measurements for multiple compounds across dozens of strains within each of the three laboratories. Overall, there are distinct differences in phenotype between manual and automated approaches for some strains and, especially, for some compounds. Lab to lab variation can also be a challenge. Nevertheless, the automated approach provides a great deal of value to the workflow of the CITP, and we plan to incorporate it into our future work, coupled with verification via manual lifespan assays.