William R. Schafer (2005) WormBook "Egg-laying."

William R. Schafer

[WormBook, 2005]

C. elegans hermaphrodites are self-fertile, and their rate and temporal pattern of egg-laying are modulated by diverse environmental cues. Egg-laying behavior has served as an important phenotypic assay for the genetic dissection of neuronal signal transduction mechanisms. This chapter reviews our current understanding of the neuronal and neurochemical mechanisms underlying the control of egg-laying in C. elegans. The roles of specific neurons in the egg-laying motor circuit, which release multiple neurotramsmitters affecting distinct parameters of egg-laying muscle activity, and the possible mechanisms for sensory control of egg-laying behavior, are discussed.

William R. Schafer (2006) WormBook "Neurophysiological methods in C. elegans: an introduction."

William R. Schafer

[WormBook, 2006]

The simple and well-defined structure of the C. elegans nervous system has made it an attractive model for studying the neural and genetic basis of behavior. However, the wider use physiological methods for monitoring neural activity in vivo or determining the effects of specific ion channels on neuronal function has been a relatively recent development. This chapter presents a compendium of protocols and technical reports on the current state of the art in C. elegans electrophysiology and neuroimaging. These include methods for calcium imaging in intact animals, in situ electrical recording from neurons and muscle cells, and in vitro recording from cultured neurons and oocytes.

Branicky, Robyn et al. (2011) International Worm Meeting "Genetic, pharmacological and calcium imaging analysis of mutants affecting HSN activity."

Schafer, William R., Branicky, Robyn

[International Worm Meeting, 2011]

We are using the egg-laying system as a model to investigate how neural circuits generate behavioural outputs. The egg-laying behaviour is regulated by a simple motor circuit, made up of only a few neurons (the HSNs and the VCs) and muscles (the VMs). Despite the anatomic simplicity of the egg-laying system, genetic, pharmacological, and more recently, calcium imaging studies have revealed several layers of complexity including the involvement of multiple neurotransmitters and their receptors as well as neuropeptides. We have used calcium imaging to monitor the activity of the HSNs and VMs in a collection of characterised and uncharacterised egg-laying defective (Egl) mutants with presumed defects in HSN activity. We have identified mutants acting both upstream and downstream of calcium entry into the HSN. We will present our phenotypic and molecular characterisation of these mutants and propose models for how they could be affecting HSN activity and ultimately the activity of the egg-laying circuit.

Branicky, Robyn et al. (2013) International Worm Meeting "The voltage-gated chloride channels encoded by clh-3 regulate the excitability of the HSN neurons."

Branicky, Robyn, Schafer, William R., Miyazaki, Hiroaki, Strange, Kevin

[International Worm Meeting, 2013]

clh-3 encodes two hyperpolarisation-activated, inwardly-rectifying chloride channel variants, CLH-3a and CLH-3b. Although the properties of these channels as well as those of their mammalian homologue CLC-2 have been well characterised, their function in neurons has not been elucidated. CLC-2 has been proposed to regulate neuronal excitability indirectly by providing a chloride efflux pathway that is necessary for GABAA-receptor mediated inhibition, or alternatively to regulate excitability directly, via a chloride influx that inhibits depolarisation. We have identified a role for clh-3-encoded channels in regulating the excitability of the HSN neurons. A gain-of-function mutation that alters the voltage sensitivity of the channels inhibits the egg-laying behaviour of C. elegans by suppressing HSN activity. Loss-of-function mutations lead to hyperactive egg-laying, including inappropriate egg-laying in the absence of food, indicating that CLH-3 channels are required for modulating the activity of the HSNs. CLH-3 channels are not required for GABAA-receptor mediated inhibition of the HSN, and do not appear to mediate chloride efflux. Rather, optogenetic and genetic epistasis experiments indicate that these channels inhibit the excitability of the HSN directly by promoting chloride influx. Thus, this family of channels could act generally to inhibit neuronal excitability by providing a pathway for chloride influx.

Kearn J et al. (2013) Invert Neurosci "Meeting report: 2012 Caenorhabditis elegans Neurobiology meeting, EMBL Advanced Training Centre, Germany."

Dalliere N, Dillon J, Kearn J

[Invert Neurosci, 2013]

Some of the finest minds in the field of Caenorhabditis elegans neurobiology were brought together from 14 June to 17 June 2012 in the small, quaint and picturesque German city of Heidelberg for the biannual C. elegans neurobiology conference. Held at the EMBL Advanced Training Centre and wonderfully organised by Diah Yulianti, Jean-Louis Bessereau, Gert Jansen and William Schafer, the meeting contained 62 verbal presentations and hundreds of posters that were displayed around the double-helical walkways that looped throughout the conference centre. Presentations on recent advances in microfluidics, cell ablation and targeted gene expression exemplified the strengths of C. elegans as a model organism, with these advances allowing detailed high-throughput analysis and study. Interesting behaviours that were previously poorly characterised were widely discussed, as were the advantages of C. elegans as a model for neurodevelopment and neurodegeneration and the investigation of neuropeptide function. The examples discussed in this meeting report seek to illustrate the breadth and depth of presentations given on these recurring topics.

Cregg, James F. et al. (2009) International Worm Meeting "Dissecting the role of serotonin and acetylcholine on egg-laying motor synapses by calcium imaging."

Schafer, William R., Cregg, James F., Chokshi, Trushal V., Chronis, Nikos

[International Worm Meeting, 2009]

Egg-laying is a simple motor program whose regulation integrates responses to diverse sensory cues (food, touch and osmolarity). The principal egg-laying motorneurons (the HSNs) use at least two neurotransmitters, acetylcholine (ACh) and serotonin (5-HT), both of which have been shown to have stimulatory and inhibitory effects on egg-laying. We attempt to tease apart the contributions of these molecules at egg-laying neuromuscular synapses by observing of the effects of exogenous pharmacological agents on both intact and dissected worm preparations with calcium imaging. We have designed a microfluidic chip that allows us to rapidly apply neurotransmitters and other pharmacological agents to intact and dissected worms. Using this approach, we have observed rapid activation of vulval muscle (VM2s) calcium transients by acetylcholine and cholinergic agonists. Indicating that acetylcholine is indeed a potent excitatory neurotransmitter at vulval muscle synapses. We have also observed more sustained activation of muscle activity by serotonin, both in intact and dissected worms. Utilizing the previously characterized serotonin receptor mutants, mod-1, ser-1, ser-4, ser-7, and the newly discovered ser-5 (generously provided by the Komuniecki lab), we have created a collection of strains containing only a single functional receptor subtype, allowing us to assess the contribution of each receptor to vulval muscle modulation by serotonin. Each serotonin receptor confers a unique response in the VM2s to different concentration of 5-HT. One, ser-4, previously characterized as inhibitory, stimulates calcium responses at concentrations of 5-HT three orders of magnitude less that which is commonly used in egg-laying assays. Other receptors display activity at significantly higher concentrations of 5-HT, closer to conditions in previous assays. Loss of any one of the five receptors causes significant loss of sensitivity to 5-HT. Further analysis of receptor phenotypes under varied conditions and in different combinations will be presented.

Galindo-Malagn XA et al. (2022) Zootaxa "New species, synonymies and records in the genus Rhagovelia Mayr, 1865 (Hemiptera: Heteroptera: Veliidae) from Colombia."

Moreira FFF, Morales I, Mondragn-F SP, Galindo-Malagn XA

[Zootaxa, 2022]

Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.

Kaulich, Eva et al. (2021) International Worm Meeting "Characterization of C. elegans acid-sensing DEG/ENaCs and their role in rhythmic behavior"

Ackley, Brian D., Schafer, William R., Kaulich, Eva, Tang, Yi-Quan, Walker, Denise S.

[International Worm Meeting, 2021]

While the roles of classical neurotransmitters and neuromodulators in neurotransmission are well-established, less attention has been paid to the effect of unconventional signaling molecules such as protons on neuronal signaling. In C. elegans, protons secreted from the intestine are able to generate motor contractions of the defecation motor program (DMP) independent of the nervous system (Beg et al., 2008). Proton-receptors from the DEG/ENaC family; called acid-sensing ion channels (ASICs) have been described in vertebrates, but their roles in behavior are mostly uncharacterized. We have been investigating C. elegans acid-sensing DEG/ENaCs and their role in modulating behavior via neuronal and non-neuronal tissues. In an initial screen using heterologous expression in Xenopus oocytes and two-electrode voltage recording, we identified two groups of C. elegans pH-sensitive DEG/ENaCs based on their response to neutral and low pH. One group, including ACD-5, DEL-4 and the previously characterized ACD-1, is open at neutral pH and blocked at low pH, while the other group, including ASIC-1, ACD-2 and DEL-9, becomes activated at low pH. The proton-inactivated channel ACD-5 is highly expressed at the apical lumen in the intestine where it senses proton-fluctuation and maintains the intestinal pH. Gain-of-function mutations show a more neutral intestinal pH and prolonged DMP intervals demonstrating the importance of protons in the generation of behavior. In contrast, the proton-activated DEL-9 is expressed in neurons and muscles. In the motoneuron AVL, DEL-9 drives the execution of the DMP enteric muscle contraction, while in the vulva muscles it is responsible for the timing of egg-laying. Mutants show prolonged inactive states during the rhythmic behavior of egg-laying and an egg-retention phenotype that can be rescued. This demonstrates that proton signaling is important for diverse physiological processes and behaviors.

Ardiel, Evan L. et al. (2011) International Worm Meeting "Investigating neural coding and interacting circuits."

Schafer, William R., Rankin, Catharine H., Rabinowitch, Ithai, Giles, Andrew C., Ardiel, Evan L.

[International Worm Meeting, 2011]

ASH mediates backward locomotion in response to osmotic pressure, nose touch, high ambient oxygen, and a variety of volatile and non-volatile compounds. We used optogenetics to simulate these stimuli to better understand their perception and how this changes with experience. Using an automated multi-worm tracker to measure behaviour, we found that a short light pulse (200-ms) of low intensity blue light elicited a reversal response of about 200-mm in 80-90% animals expressing ChR2 in ASH. A longer light pulse (2-s) elicited a larger reversal response (1-mm) that often ended in an omega turn. Previous work demonstrated that ASH-mediated responses to different stimuli were genetically dissociable (Mellem et al., 2002). We monitored the responses of eat-4, glr-1, nmr-1, and glr-1;nmr-1 mutants and found that the behavioural impairments associated with nose touch and osmo-avoidance assays were also apparent in response to the short and long light pulses, respectively. This suggests that a part of the neural code distinguishing nose touch and chemoaversion is the duration of depolarization of ASH. We next tested habituation of ASH to our "nose touch" and "chemoaversive" light pulses. We found that both responses decremented from repeated exposure, but with differential kinetics. The proportion of worms responding to the longer light pulse decreased faster and further than responses to the nose touch-like stimulus. Disrupting neuropeptide signalling blocked this deep habituation. The interneurons downstream of ASH largely overlap with those mediating reversals to body touch. We were interested in addressing how activity in one circuit affected the other. We found that the tap response was decremented by a single light, but facilitated by repeated presentation of long-light pulses. We are currently investigating the cellular mechanisms underlying this cross-circuit plasticity.

Yemini, Eviatar I. et al. (2013) International Worm Meeting "A database of C. elegans behavioral phenotypes."

Brown, Andre E.X., Yemini, Eviatar I., Grundy, Laura J., Schafer, William R., Jucikas, Tadas

[International Worm Meeting, 2013]

Previous single and multi worm tracking experiments have produced summary statistics to phenotype small worm sets. We introduce a database of extensive and intensive single-worm phenotypes for over 300 strains of C. elegans with nervous system and locomotory defects as well as a reference of N2 variability composed of more than 1,200 young-adult hermaphrodites examined over the course of 3 years. The data is available online at http://wormbehavior.mrc-lmb.cam.ac.uk and includes a link to Worm Tracker 2.0 (our single-worm tracker used for data collection). Our phenomic database provides multiple levels of representation, from high-level statistical strain summaries all the way down to detailed time-series measurements for over 10,000 single-worm experiments. Within our database are 76 mutants with no previously characterized phenotype, 15 genes with multiple allelic representation, and 13 double or triple mutant combinations (the majority of which are accompanied by single mutant representation as well). Annotated experimental videos are easily accessible alongside their data, with various degrees of processing, from the skeleton and outline coordinates to the time series of extracted features, their histograms, and an in-depth view of collective strain statistics. For computational researchers, the database is a rich source of processed measures and raw data for developing new algorithms for segmentation, behavioral quantification, and bioinformatic approaches which link complex phenotypes with genetic perturbations. For neurogeneticists, the summary statistics and visualizations make it possible to identify behavioral phenotypes in mutants of interest. Free Worm Tracker 2.0 software and simple plans to build its inexpensive hardware are available at http://www.mrc-lmb.cam.ac.uk/wormtracker/.