Brownlee DJA et al. (1996) Parasitol Today "Nematode neuropeptides: Localization, isolation and functions."

Holden-Dye L, Brownlee DJA, Walker RJ, Fairweather I

[Parasitol Today, 1996]

Historically, peptidergic substances (in the form of neurosecretions) were linked to moulting in nematodes. More recently, there has been a renewal of interest in nematode neurobiology, initially triggered by studies demonstrating the localization of peptide immunoreactivities to the nervous system. Here, David Brownlee, Ian Fairweather, Lindy Holden-Dye and Robert Walker will review progress on the isolation of nematode neuropeptides and efforts to unravel their physiological actions and inactivation mechanisms. Future avenues for research are suggested and the potential exploitation of peptidergic pathways in future therapeutic strategies

Mulcahy, Ben et al. (2013) International Worm Meeting "Age-dependent changes at the C. elegans neuromuscular junction."

O'Connor, Vincent, Holden-Dye, Lindy, Mulcahy, Ben

[International Worm Meeting, 2013]

Age-dependent synaptic dysfunction is an established determinant of health span and plays a major role in neurodegenerative disease. However, mechanisms of synaptic dysfunction are poorly understood. We are using behavioural, pharmacological and electrophysiological techniques to probe the C. elegans neuromuscular junction across age. First we observe a progressive decline in motility as worms age. This could be due to altered neuromuscular transmission, muscle function, alterations upstream of the neuromuscular junction or a combination of these. To resolve this we designed pharmacological assays that can probe neuromuscular function in the absence of spontaneous movement, the latter being a confounding factor in aged worms. We used two cholinergic drugs, levamisole and aldicarb. Both cause contraction of the worms. Levamisole directly activates cholinergic receptors on muscle cells and aldicarb causes a build-up of acetylcholine (ACh) in the synaptic cleft that is first dependent on synaptic release from the motor neuron. Comparing the effects of these drugs evidenced increased release of ACh from the presynapse during early ageing, which declined during advanced age to levels similar to those seen in young worms. Using a daf-2(e1370) mutant worm we have shown that the increase in ACh release during early ageing is dependent on insulin signalling. These data also highlight that the ability of the muscle to contract is retained even in old age (Mulcahy et al., 2013). We have followed this up with patch clamp recording of mini-endplate currents from the body wall muscle to assess spontaneous neurotransmitter release across the same ageing time course. We show that the electrophysiological data correlate with the pharmacological data and substantiate an increase in neuromuscular signalling in early ageing followed by a gradual decline. It is interesting that the increased strength of neuromuscular transmission during early ageing is coincident with a period of behavioural decline. It is also interesting that in very old worms we see evidence of functional neuromuscular synapses yet the worms remain immobile. We are using patch clamp recording from the body wall muscles to follow this up further.

Crisford, Anna et al. (2013) International Worm Meeting "Leveraging C. elegans cue-dependent behaviour to understand the host/parasite interaction for plant parasitic nematodes."

Urwin, Peter E, Marvin, Jessica, Kearn, James, Holden-Dye, Lindy, O'Connor, Vincent, Lilley, Catherine, Crisford, Anna

[International Worm Meeting, 2013]

Plant parasitic nematodes (PPNs) are a global problem inflicting crop damage costing circa $125b per annum. The chemicals used to control these pests have an unacceptable environmental impact and are being withdrawn from use. This presents a challenge as without adequate crop protection the PPNs present a real threat to food security. The rationale for the study is that neurobiological underpinnings of C. elegans environmentally driven cue-dependent behaviour may be conserved in the PPNs and provide a route to new molecular targets for pest control. We have identified orthologues of C. elegans genes encoding key components of neurotransmitter signalling pathways in the potato cyst nematode Globodera pallida. We have characterised a G. pallida orthologue of C. elegans tph-1, which encodes the synthetic enzyme for 5-HT. C. elegans mutant tph-1(mg280) is defective in feeding and this was completely rescued by expression of the G. pallida orthologue from a pan neuronal promoter. Therefore this gene is most likely required for 5-HT synthesis in PPNs. In PPNs the host plant stimulates the activity of the stylet, a feeding tube that protrudes from the mouth of the worm and which is intimately involved in invasion of the roots (1). We show electrophysiological recordings from G. pallida provide a readout of stylet thrusts and we are currently investigating host plant cues in this paradigm. This will dovetail with RNAi experiments for tph-1 to delineate the role of 5-HT in host cue-dependent behaviour. Funded by the Biotechnology and Biological Sciences Research Council UK Grant no BB/K012495/1. We gratefully acknowledge CGC for provision of tph-1(mg280). 1.Holden-Dye, L., and Walker, R. (2011) Neurobiology of plant parasitic nematodes. Invertebrate Neuroscience, 1-11.

Mulcahy, Ben et al. (2013) International Worm Meeting "Does Cysteine String Protein contribute to C. elegans Nervous System Function?"

Holden-Dye, Lindy, Ibbett, Paul, O'Connor, Vincent, Mulcahy, Ben

[International Worm Meeting, 2013]

Cysteine string protein (CSP) is a synaptic vesicle protein that uses its DnaJ domain to co-chaperone Hsc70 allowing it to refold SNAP-25 and other synaptic proteins. It appears to be a significant resilience factor at synapses as in Drosophila and mice, mutations in CSP result in synaptic dysfunction, neurodegeneration and early death. We have shown that both ageing and insulin signalling modulate synaptic function at the C. elegans body wall neuromuscular junction (Mulcahy et al., 2013), and wanted to see if we could model accelerated synaptic dysfunction with age using mutants deficient in the worm orthologue of CSP, dnj-14. Initial tests with these strains indicated that in contrast to the phenotypes seen in Drosophila and mice, there was not a decreased lifespan compared to wild-type. Therefore we repeated the lifespan assays after repeated backcrossing (6x) into our laboratory wild-type strain. There was no effect of CSP mutation on lifespan, even at elevated temperatures. Behavioural correlates of synaptic function are not defective when compared to wild-type, even across ageing. In addition, pharmacological assays argue against accelerated synaptic dysfunction across ageing in this genetic background. This is surprising considering the severity of the phenotypes seen in Drosophila and mouse CSP mutants. Electrophysiological recordings from the body wall muscle are currently underway and provide another route to identify altered neuronal signalling either at the neuromuscular junction itself or upstream in the neural network that regulates its activity. We are also extending our analysis into assays that require sensory integration in order to conclusively test if the worm CSP gene is required for the maintenance of intact behaviours across ageing in C. elegans.

Hitchcock DR et al. (1998) Arch Environ Contam Toxicol "Determining toxicity trends in the ozonation of synthetic dye wastewaters using the nematode Caenorhabditis elegans."

Law SE, Hitchcock DR, Williams PL, Wu JH

[Arch Environ Contam Toxicol, 1998]

The nematode Caenorhabditis elegans was used in 72-h toxicity tests to evaluate the influence of ozonation on the toxicity of three synthetic azo dye wastewaters (two reactive dyes and one acid-based dye). The two reactive dye wastewaters contained high concentrations of NaCl (89-112 g/L) in addition to potentially toxic dye components. To determine the contribution of NaCl to toxicity, simulated dye wastewater samples with and without NaCl were tested. Samples were collected at various times during ozonation (t = 0, 8, 32, 64 min); nematodes were exposed to the samples for 72 h. The influence of ozonation on toxicity varied between dye wastewater types. For the acid-based dye wastewater, toxicity increased as duration of ozonation increased. For the reactive dyes without NaCl, toxicity did not appear to be influenced by ozonation. For the reactive dyes with NaCl, mortality was 100% with or without ozonation. Range-finding experiments with NaCl in water and NaCl in dye wastewaters suggested an additive toxic interaction between NaCl and the dyes in wastewater to the nematodes. The duration of ozonation for acid-based dyes and the relatively high NaCl concentrations for the reactive dyes appear to influence effluent toxicity in the ozonated dye wastewaters.

Tong YG et al. (2010) J Neurosci Methods "Conditions for dye-filling of sensory neurons in Caenorhabditis elegans."

Burglin TR, Tong YG

[J Neurosci Methods, 2010]

Dye-filling is a common method used to stain Caenorhabditis elegans sensory neurons in vivo. While the amphids and phasmids are easy to stain, a subset of sensory neurons, the IL2 neurons, are difficult to stain reproducibly. Here we examined the conditions under which the IL2 neurons take up the lipophilic fluorescent dye DiI. We find that IL2 dye-filling depends on salt concentration, but not osmolarity. Low salt prior and during incubation is important for efficient dye uptake. Additional parameters that affect dye-filling are the speed of shaking during incubation and the addition of detergents. Our modified dye-filling procedure provides a reliable method to distinguish mutant alleles that stain amphids and phasmids, IL2 neurons, or both. An additional benefit is that it can also stain the excretory duct. The method allows genetic screens to be performed to identify mutants that selectively affect only one of the sensory structures or the excretory duct.

Carrel JS (1976) Worm Breeder's Gazette

Carrel JS

[Worm Breeder's Gazette, 1976]

Unfertilized oocytes, 'squashy eggs', can be scored on a petri plate by adding a few drops of 0.05% Trypan blue to the surface of the plate. The oocytes take up the dye and worms and zygotes do not. The dye has no apparent effect on the worms left on the plate.

Caroline Craig et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "The neuropeptide receptor NPR-2 modulates cholinergic release and is important in locomotory and egg laying behaviors"

William Schafer, James Cregg, Caroline Craig

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Neuropeptide signaling is widely posited to influence addiction behaviors, perhaps the best-known example being the role of corticotropin-releasing factor (CRF) in drug dependency and relapse. In a RNAi screen for genes important for nicotine resistance, we identified neuropeptide receptor 2 (npr-2). Based on analysis of npr-2 mutants (kindly provided by N. Kriek and L. Holden-Dye), we have identified defects in both locomotory and egg laying behaviors. In addition, responses to both nicotine and levamisole are altered. These phenotypes suggest that NPR-2 may modulate acetylcholine release in both neural circuits. We are currently attempting to address this question using neural imaging techniques.

Dillon, James C et al. (2009) International Worm Meeting "Refinement of the concentration-dependent effects of ethanol on the behaviour of the C. elegans pharynx."

Mould, Richard, James, Christopher, Andrianakis, Ioannis, Holden-Dye, Lindy, O'Connor, Vincent, Dillon, James C

[International Worm Meeting, 2009]

The effects of ethanol upon the mammalian brain are broad ranging and concentration-dependent. Low concentrations (17mM) intoxicate while greater than 65mM anaesthetize. The responses to ethanol involves multiple substrates, which include receptors, ion channels, intracellular signalling molecules and changes to the properties of membranes. The concentration-dependent effect of ethanol on C. elegans remains unclear due to conflicting reports of ethanol''s equilibration across the worm cuticle. This compromises our understanding of C. elegans as a relevant model for human ethanol intoxication and dependence and for the identification of the underlying neuronal substrates. To further clarify the concentration-dependent effects of ethanol upon the C. elegans nervous system we have made electrophysiological recordings from the pharynx (electropharyngeograms (EPG)) in the presence of a range of ethanol concentrations (10-300mM). The EPG provides a readout of muscle activity driven by the pharyngeal nervous system. Recordings were made from a semi-intact preparation of the pharynx which allows ethanol direct access to the pharyngeal muscle and nervous system, thus the concentration acting on this tissue can be clearly defined. We have designed software that facilitates the semi-automated statistical analysis of EPG recordings, enabling the identification and comparison of discrete pharyngeal phenotypes. Using this software we have identified ethanol concentration-dependent effects on the behaviour of the pharynx. At 10mM, a concentration close to human intoxication ethanol caused a subtle change in the pattern of pharyngeal activity stimulating worms to pump in larger ''bursts''. 50mM ethanol caused a change in the shape of the EPG recording corresponding to muscle depolarization. We have identified the threshold for this effect is close to 50mM and that it becomes more pronounced as the concentration of ethanol increases. At 300mM, ethanol caused a rapid, transient excitation of pharyngeal pumping followed by a rapid inhibition. We are further investigating the effects of ethanol on the pharynx by performing intracellular recordings and video analysis of muscle behaviour to elucidate the nature of the electrophysiological changes observed at 50mM. It is intended that this will provide further insight into the molecular substrates and cellular mechanisms underpinning the acute and chronic effects of ethanol in the mammalian system.

Jardim NS et al. (2024) J Vis Exp "Using Low-cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans."

Silva AC, Avila DS, Jardim NS

[J Vis Exp, 2024]

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.