Flavell, Steven W et al. (2013) International Worm Meeting "Serotonin and PDF are opposing neuromodulators that control a bistable foraging behavior in C. elegans."

Pokala, Navin, Albrecht, Dirk A, Macosko, Evan Z, Bargmann, Cornelia I, Larsch, Johannes, Flavell, Steven W

[International Worm Meeting, 2013]

Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete, long-lasting behavioral states. Here we characterize a neuromodulatory circuit that generates such long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces long-lasting dwelling states. PDF promotes roaming states through the Gas-coupled PDFR-1 receptor; optogenetic activation of cAMP production in PDFR-1-expressing cells induces long-lasting roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of the neuromodulatory circuit supplement fast motor circuits to give rise to long-lasting behavioral states.

Catharine Rankin et al. (2003) International Worm Meeting "The effects of avr-14 on short- and long-term memory."

Nicholas Dube, Stephan Steidl, Catharine Rankin

[International Worm Meeting, 2003]

avr-14 encodes an -type subunit of a glutamate-gated chloride channel (GluCls) in Caenorhabditis elegans. avr-14 is expressed on the glutamatergic mechanosensory neurons of the tap-withdrawal response that synapse onto the command interneurons of the circuit. Because avr-14 is expressed on the touch cells it was hypothesized that it might play a role in experience-dependant plasticity in the response to tap. Worms with a mutation in avr-14 were tested for short-term and long-term habituation of the tap response. The mutation selectively affects short-term habituation for shorter interstimulus intervals (ISIs; 10s and 30s) while not affecting habituation for long ISIs (45s and 60s). These results provide support for the hypothesis that in C. elegans there are multiple ISI-dependant short-term memory systems. avr-14 worms show long-term memory for habituation training comparable to wild-type controls when given distributed training and testing with a 60s ISI. avr-14 worms, unlike wild-type controls, also show long-term memory when trained and tested with a 10s ISI. Furthermore, long-term memory for this training protocol is protein synthesis dependant, as administering heat shock in between blocks of training blocks the formation of long-term memory. This suggests that in wild-type worms the presence of avr-14 blocks the conversion from short-term to long-term memory following training with short ISIs.Thanks to Joe Dent for avr-14.

Li, Runsheng et al. (2015) International Worm Meeting "Illumina Synthetic Long Read Sequencing Allows Recovery of Missing Sequences even in the "Finished" C. elegans Genome."

Zhao, Zhongying, Li, Runsheng, Young, Amanda, Zhang, Zhihong, Ren, Xiaoliang, Hsieh, Chia-Ling

[International Worm Meeting, 2015]

Next-Generation Sequencing permits rapid acquisition of high-throughput DNA sequences, but its reads are relatively short in length, limiting their use in genome-based studies. Illumina has recently released a technology called Synthetic Long-Read Sequencing that can produce reads of unusual length, i.e., predominately around 10 Kb. However, a systematic assessment of their use in genome finishing and assembly is still lacking, especially in resolving the repetitive sequences. We evaluate the promise and deficiency of the long reads in these aspects using C. elegans genome. First, the reads are highly accurate and capable of recovering most types of repetitive sequences. However, the presence of tandem repetitive sequences that extend over certain length prevents pre-assembly of the long reads inside this genomic region. Second, the reads can reliably recover the missing but not the extra sequences in C. elegans genome. 24X of the long reads allow the recovery of at least 40 Kb of missing genomic DNAs that are located inside or outside coding region. Finally, an N50 size of at least 86 Kb can be achieved for the contigs that are de novo assembled with the 24X reads. However, substantial mis-assembly errors are observed which are caused by either mis-assembled long read or flanking repetitive sequences, highlighting a need for novel assembly algorithm to accommodate the long reads in de novo genome assembly. The long reads are expected to be useful in generating a "finished-grade" genome of other nematode species if coupled with independent data such as those from PacBio or mate-pair sequencing or repairing the existing nematode draft genomes on its own.

Steven J Husson et al. (2009) European Worm Neurobiology Meeting "Optogenetics tools to dissect a nociceptive neuronal network and neuropeptide signalling pathways in C. elegans"

Alexander Gottschalk, Jana Liewald, Steven J Husson, Liliane Schoofs, Martin Brauner, Wagner Steuer Costa

[European Worm Neurobiology Meeting, 2009]

Light-gated cation channels such as the blue light-activated depolarizing Channelrhodopsin-2 (ChR2), allow optical activation of individual neurons of live and behaving C. elegans at the millisecond time-scale in a non-invasive manner (Nagel et al., 2005, Zhang et al., 2007, Liewald et al., 2008). This optogenetics approach paves the way for further functional dissection of peptide signalling pathways or individual neuronal networks in a detail that is not possible in higher organisms. The huge advantage is that we can specifically stimulate the sensory input neurons, while other potentially contributing neurons are kept silent. The sensory PVD neurons that envelop the nematode with highly branched dendritic arbors are involved in harsh touch nociception. Expression and activation of ChR2 in PVD results in a forward escape movement and sometimes a reversal. These results are in line with the fact that the PVD neurons make synaptic contacts with the locomotory command interneurons PVC and AVA that regulate forward and backward movement, respectively. Using electrophysiology, we will assess the physiology of the PVD cells, as well as the downstream interneurons in response to photoactivation, while the involvement of different ion channels, receptors or neurotransmitters will be assessed by RNAi. We are also investigating the flp-15 and nlp-38 neuropeptidergic signalling pathways by optogenetics. Neuropeptide release can be triggered by photo-activating the respective neurons in an acute fashion while effects on behaviour can be observed at the same time. This way, we can correlate neuropeptide action with acute behavioural changes or effects, about which very limited knowledge is currently available in any system. References Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, and Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods, 5, 895-902. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, and Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol, 15, 2279-2284. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, and Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature, 446, 633-639.

Suzannah Hetherington et al. (2005) International Worm Meeting "Functional Analysis of gei-16, a Developmental Gene with Multiple Splice Variants"

Nick Johnson, Carolyn Behm, Flavia Pellerone, Suzannah Hetherington

[International Worm Meeting, 2005]

The gene gei-16 was originally identified as the C. elegans homolog of OvB20, an antigen expressed by the parasitic nematode Onchocerca volvulus. It has since been shown to have a role in embryonic elongation . This gene appears to be nematode-specific, with no clear homolog in humans, mice or flies but clear matches to a range of parasitic nematodes. gei-16 appears to have at least 19 different splice variants in C. elegans. These can be divided into three major types: long variants, short variants and the d variant. Knockdown by RNAi of all variants in N2 shows a severe phenotype with a high penetrance of Adl, Prz, Unc, Clr and Lva additional to the published Emb, Lva , Gro and Lva phenotypes. RNAi was directed against unique regions of the short and long variants. No obvious phenotype was seen for the short variants, however, knockdown of the long variants produced a severe phenotype identical to that found for knockdown of all variants. This may suggest that the long variants is most important for development. Semi-quantitative RT-PCR shows the long transcript is expressed through all stages of development, but is most highly expressed in the embryonic and early larval stages. Long variant expression levels appear higher than short variant levels in all stages. A GFP reporter construct is currently being made to elucidate the spatial and temporal expression of gei-16.

Alexander Mendenhall et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Uncoupling Mechanisms Required for Oxygen Deprivation Survival and Longevity in C. elegans"

Michelle LeBlanc, Alexander Mendenhall, Pamela Padilla, Mary Ladage, Mark Pavlyukovskyy

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Stress resistance and longevity occur from single gene mutations, suggesting that these two traits are regulated by similar mechanisms. Others have shown that mutations in the insulin-like signaling pathway (daf-2(e1370)) or alterations that affect germline (glp-1(q158)) lead to increased lifespan. We show that these same mutations lead to severe oxygen deprivation survival (anoxia), indicating that stress resistance and longevity are under control of DAF-16 or signals involving the reproductive tissue. Further analysis of mutations that enhance long-term anoxia survival indicates that there are distinct genetic and physiological factors affecting anoxia responses that do not affect longevity. For example, the DAF-16-regulated glycolytic genes, gpd-2/3, are required for the daf-2(e1370) animals'' long-term anoxia and high temperature anoxia survival phenotype but not the longevity phenotype. Furthermore, we have identified mutants with reproductive phenotypes, such as the fog-2(q71) animals, which have an enhanced ability to survive long-term anoxia but are known to not be long lived. Additionally, not all daf-2 mutants that are long lived will survive long-term anoxia. Thus, data suggests that modulation of distinct components of the insulin signaling pathway or specific changes in reproductive physiology differentially regulate anoxia survival and longevity. Therefore, we hypothesize that the mechanisms required for anoxia stress responses and longevity are distinct and can be uncoupled. To test this hypothesis we are conducting further analysis on the functional role of gpd-2/3. First, we are determining if gpd-2/3 function is required for other long-term anoxia survival mutants. Second, we are using a transcriptional reporter (Pgpd-2/3::GFP) to analyze gpd-2/3 expression in anoxia exposed animals, aging adults or daf-16(mu86) null mutants. Finally, we tested whether transgenic over expression of GPD-2/3 is sufficient to increase long-term anoxia survival or enhance lifespan. This research will further our understanding of the relationship between oxygen deprivation resistance and longevity.

Jacqueline Rose et al. (2001) International C. elegans Meeting "Long-Term Habituation is Dependent of Both Glutamate Availability and AMPA Receptor Function in C. elegans."

Catharine Rankin, Jacqueline Rose, Karla Kaun, Sylvia Chen

[International C. elegans Meeting, 2001]

C. elegans is capable of showing long-term memory for habituation training of the tap withdrawal response. As in other organisms this training is best produced by distributed or spaced training and lasts at least 24 hours. The neural circuit underlying the tap withdrawal response has been described and there is increasing evidence that the sensory neurons use glutamate as their neurotransmitter (Rankin and Wicks, 2000). Long-term habituation training for the current experiments consisted of groups of approximately 20 worms that received 4 blocks of 20 taps at a 60s interstimulus interval (ISI) with blocks separated by one-hour rest periods. Testing was conducted 24 hours after training with individual worms given one block of 10 taps also at a 60s ISI. Trained groups were compared to a control group that received a single tap on training day and the same individual testing on day two. The results showed that wild-type worms that received distributed training show significantly smaller responses compared to single-tap controls (p<0.01). This retention has been observed at 24, 48 and 72 hours following training. eat-4 (ky5) mutants also received distributed habituation training and although previous studies have shown that eat-4 (ky5) mutants display enhanced short-term habituation the eat-4 (ky5) mutant did not show long-term memory for the habituation training. In a follow-up study, eat-4 (ky5) mutants were given the same distributed habituation training protocol but using a stronger stimulus (trains of 7 taps/sec) rather than the single tap. This resulted in trained eat-4 (ky5) mutant worms showing decreased reversal magnitude on Day 2 compared to untrained eat-4 (ky5) controls. This suggests that stronger stimulation likely results in increased glutamate release from the sensory neuron in these mutants. The alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)-receptor subtype mutant glr-1 (n2461) is capable of short-term habituation, however, when long-term habituation training was administered to the glr-1 (n2461) mutant this resulted in no long-term retention of the training on Day 2. When glr-1 (n2461) worms were given the stronger stimulation (trains of taps) they still did not show long-term habituation. Thus the AMPA receptor does appear to be necessary for long-term memory regardless of level of stimulation. To further elucidate the role of glutamate in long-term memory wild-type worms were placed on plates streaked with either 10 mM 6,7-dinitrquinoxaline-2,3-dione (DNQX; a competitive AMPA-receptor blocker) or vehicle (35 mM sodium hydroxide; NaOH) during training on Day 1. Day 2 testing was conducted drug free. The vehicle-administered worms displayed long-term retention while the worm exposed to DNQX did not. This finding, coupled with the long-term memory deficits observed in the glr-1 mutant strain, lead to the conclusion that activation of the glutamate receptor subtype AMPA is required for the formation of long-term retention. The role of the N-methyl-D-aspartate (NMDA) glutamate receptor in long-term habituation training was also investigated. Wild-type worms were trained with the non-competitive NMDA antagonists (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK801). When worms were trained in the presence of MK801 there was no evidence for long-term memory of habituation training. This supports what has been theorized in mammalian models of long-term synaptic plasticity in that activation of AMPA-type receptors may be required for NMDA-type receptor activation and therefore when either receptor is blocked, it effectively attenuates long-term memory. Research funded by the Natural Sciences and Engineering Research Council of Canada.

Smith, R.L. et al. (2017) International Worm Meeting "Metabolomics and fluxomics reveal a metabolic fingerprint of the long-lived mutants daf-2 and eat-2."

Houtkooper, R.H., van Weeghel, M., Gao, A.W., Smith, R.L.

[International Worm Meeting, 2017]

The revival of metabolism as a major focus in healthcare and ageing research has yielded many new techniques and insights. We developed and applied state-of-the-art metabolomics techniques on the Q-Exactive platform to analyze the long-lived mutants daf-2 and eat-2 in search of a metabolic fingerprint of ageing. These mutants show reduced concentrations of many metabolites including amino acids, nucleotides, and pentose phosphate pathway (PPP) intermediates. Of note, thymidine monophosphate, NAD+, and NADH were higher in long-lived mutants compared to controls. To gain more insight into the metabolic control of these mutants we analyzed their transcriptome. Long-lived mutants both show down-regulation of amino sugar and nucleotide sugar metabolism, glycosaminoglycan biosynthesis, and folate synthesis. They also both show up-regulation of ascorbate and aldarate metabolism, drug and xenobiotic metabolism, glutathione metabolism, glycine, serine and threonine metabolism, retinol metabolism, and branched chain amino acid biosynthesis. Using Pathview in 'R', we combine both metabolic and transcriptomic data, extracting relevant changes in the metabolome of the long-lived mutants based on the expression of genes annotated in glycolysis, TCA cycle, PPP, nucleotide, and amino acid pathways. We find that glycolysis and the PPP are similarly regulated in the long-lived mutants and are possibly major pathways related to longevity in these animals. To investigate substrate preference and metabolic flexibility in these long-lived animals we initiated tracer-based metabolomics using the stable isotope universally-13C-labeled glucose. To calculate metabolic fluxes, we use the incorporation of 13C atoms in metabolites to perform mass isotopomer distribution analysis. We conclude that long-lived mutants have a distinct metabolic regulation that likely underlies their increased longevity and that metabolomics methods in C. elegans are ready to be utilized to investigate complex health-related traits.

Thatcher, Elizabeth J et al. (2013) International Worm Meeting "Acute, High-throughput RNAi in C. elegans."

Ambros, Victor, Thatcher, Elizabeth J

[International Worm Meeting, 2013]

C. elegans have previously demonstrated a robust ability to utilize exogenous dsRNA to silence endogenous genes and transgenes by RNA interference (RNAi). Typically, dsRNA is delivered for RNAi by microinjection, soaking in naked dsRNA, or feeding of bacteria expressing dsRNA. Microinjection requires training and expensive equipment and is usually applied to relatively small numbers of worms. Soaking and feeding are more amenable to higher throughput, although soaking requires a larger quantity of dsRNA, and both feeding and soaking can involve a relatively long time course between application of the dsRNA and when knockdown is observed. We have developed a method for the use of electroporation to efficiently deliver dsRNA to C. elegans larvae or adults for silencing of somatic or germline genes. Electroporation offers a number of significant advantages over previous methods for RNAi in C. elegans. Electroporation results in rapid delivery of dsRNA to populations of worms without expensive equipment or training. Electroporation requires a lower concentration of dsRNA than does soaking, and results in more rapid knockdown than does feeding or soaking. Electroporation is most effective at long-term knockdown when using long dsRNA rather than short (25 base pairs) dicer substrate RNA or siRNA (22 base pairs, with 2 nt 3' overhangs). Interestingly, short dsRNAs and siRNAs result in acute but transient knockdown (for example of a GFP transgene), whereas long dsRNA results in long-term (and heritable) knockdown. Long dsRNA can be easily in vitro transcribed from widely available RNAi plasmids. Electroporation can also be completed using dsRNA extracted from bacteria expressing RNAi plasmids. This allows for efficient screening of RNAi libraries using a 96-well format. Thus, electroporation can be easily employed for rapid and either transient or long-term knockdown of endogenous genes or transgenes on a high-throughput scale.

McEwan, Andrea et al. (2011) International Worm Meeting "Candidate screen for mutants defective in long-term memory of habituation."

McEwan, Andrea, Rankin, Catharine

[International Worm Meeting, 2011]

Neurodegenerative-dependent memory loss is becoming increasingly relevant in today's aging population. The mechanisms that underlie memory are complex and, as yet, not fully understood. C.elegans offers a unique opportunity to study the biochemical pathways central to learning and memory. C.elegans has a simple, fully mapped nervous system and demonstrates several types of memory, including long-term memory for habituation. In the past, researchers have shied away from behavioural screens for long-term memory for habituation because of the labor intensive and time consuming methods used for data collection and analyses. Recently, a fully automated system called the multi-worm tracker has been developed and used to investigate genes involved in short-term habituation. The multi-worm tracker is able to record and analyze the movement of individual worms within a population in real time, greatly increasing the efficiency of behavioural studies. We have established a new protocol for investigating long-term memory for habituation on the multi-worm tracker and plan to perform a high-throughput candidate screen for mutants that fail to show long-term memory of habituation to mechanosensory stimulation. A screen for long-term memory for habituation could potentially identify novel players involved in aspects of memory, including genes involved in memory formation and memory storage.