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Purpose

C. elegans is a well-validated experimental subject for toxicological research¹ including the physiological effects of specific toxic substances and genes that influence sensitivity or resistance to these substances. C. elegans also provides a whole-animal system for assaying the toxicity of environmental soil or water samples. The U.S. National Institute of Environmental Health Sciences has highlighted C. elegans as a valuable system for toxicological research².

The NemaMetrix ScreenChip System is a microfluidics platform for recording electropharyngeograms (EPGs) from the nematode worm Caenorhabditis elegans and other species. Various endpoints have been used to detect toxic effects, including development, reproduction, induction of stress-related genes, lethality and—relevant to the ScreenChip system—pharyngeal pumping. Toxicants that inhibit pumping in C. elegans include heavy metals, insecticides, organophosphate pesticides and cyanobacterial toxins. Here we used EPG recordings to investigate toxic effects of the heavy metal, copper (Cu²⁺), which inhibits pharyngeal pumping³. Exposure to high levels of Cu²⁺, e.g., from corrosion of copper pipes by acidic water, is likewise toxic to humans and can damage the liver and kidneys⁴.

We found that microfluidic EPG recordings detected quantitatively the presence of Cu²⁺ in aqueous samples at concentrations within the range found in contaminated home water supplies.

Results and Discussion

Experiment 1 was performed using 8-channel microfluidic EPG chips with perfusion capability^5,6. Fig. 1A shows pharyngeal pump frequency in control
solution (K medium + 10 mM 5HT), followed att = 0 min by switching the perfusate to the same solution containing a range of Cu²⁺ concentrations. In control worms (black line), pump frequency remained steady over time, at ~ 4 Hz (pumps/s). In response to Cu²⁺ exposure, pump frequency decreased in a concentration–dependent manner, with inhibition apparent within 5 min at the higher concentrations. Steady-state pump frequency, defined as the mean frequency between t = 30 and 60 min, was plotted in Fig. 1B to derive an IC₅₀ value (the concentration that caused a 50% reduction in pump frequency) of 49 mg/L Cu²⁺. For comparison, a prior study reported an IC₅₀ of 3.32 mg/L Cu²⁺ when pumping was counted visually after 24 h exposure on Cu²⁺-containing agar plates³. Thus—not unexpectedly—Cu²⁺-induced inhibition of pumping depends on both the concentration and duration of exposure.

Experiment 2. In these experiments, the ScreenChip system was used to compare pump frequency in worms exposed to control or 50 mg/L Cu²⁺ solutions (Fig. 2; n = 26-28 worms/group; mean ± S.E.M.). EPG recordings (2 min per worm) were started 30 min after the onset of Cu²⁺ exposure, during the steady-state inhibition of pumping (see Fig. 1A). The Cu²⁺-exposed group showed a significant reduction in pump frequency of ~68% compared to controls (P < 0.00001; 2-tailed Mann-Whitney Wilcoxon Test).

Conclusions

These data demonstrate the use of microfluidic EPG recordings to quantify concentration- and time-dependent effects of Cu²⁺ on pharyngeal pumping. To our knowledge, this is the first demonstration of a rapid, electrophysiological effect of Cu²⁺ on C. elegans. EPG recordings detected [Cu²⁺] as low as 10 mg/L (Fig. 1), well within the range of copper-contaminated water in the United States, which can exceed 30 mg/L⁴; for comparison, the U.S. Environmental Protection Agency’s upper limit for safe drinking water is 1.3 mg/L Cu²⁺. We conclude that the ScreenChip system provides rapid and sensitive detection of the toxic heavy metal, Cu²⁺, in a concentration range suitable for testing environmental samples.

Methods

Synchronized N2 (wild-type) worms were cultivated at 20 ^oC to the first day of adulthood on plates containing nematode growth medium (NGM) seeded with E. coli OP50, using standard methods^7,8. For microfluidic EPG recordings, reagent-grade Cu²⁺ solutions were prepared in K medium (32 mM KCl, 51 mM NaCl in dH₂O) containing 10 mM 5HT to stimulate pumping^5,6,9. EPG recordings were acquired using Spike2 software (Cambridge Electronic Design Ltd.) for 8-channel chips or NemAcquire software¹⁰ for the ScreenChip system. Detailed ScreenChip methods are available elsewhere¹¹. Recordings were analyzed using custom software in IGOR Pro⁶, soon to be superseded by the release of NemaMetrix’s NemAnalysis software¹⁰.

 

References

1. Leung MC et al. (2008) Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology. Toxicol Sci. 106(1):5-28
2. C. elegans: a medium-throughput screening tool for toxicology (2006). http://ntp.niehs.nih.gov/ntp/factsheets/wormtoxfs06.pdf
3. Jiang Y et al. (2016) Sublethal toxicity endpoints of heavy metals to the nematode Caenorhabditis elegans. PLoS One, Jan 29;11(1):e0148014
4. Donohue J (2004) Copper in drinking-water: background document for development of WHO guidelines for drinking-water quality. http://www.who.int/water_sanitation_health/dwq/chemicals/copper.pdf
5. Lockery SR et al. 2012. A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans. Lab Chip 12:2211-2220
6. Weeks et al. Microfluidic platform for electrophysiological recordings from host-stage hookworm and Ascaris suum larvae: a new tool for anthelmintic research.  Int J Parasitol: Drugs Drug Res, in press
7. Stiernagle T. 2006. Maintenance of C. elegans. WormBook, http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
8. Porta-de-la-Riva M et al. 2012. Basic Caenorhabditis elegans methods: synchronization and observation J Vis Exp 64:4019
9. https://nemametrix.com/serotonin-induced-pharyngeal-pumping-c-elegans-using-screenchip/
10. https://nemametrix.com/downloads/
11. https://nemametrix.com/wp-content/uploads/2016/08/ScreenChip_System_QuickStart_Guide.pdf; https://nemametrix.com/tech-notes

Acknowledgements

Unpublished data were provided by JC Weeks, KJ Robinson and WM Roberts. Funding from Oregon BEST.

Do Nematode Worms get ‘The Munchies?’

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Purpose

Cannabinoid drugs mimic endocannabinoids, endogenous signaling molecules used by the brain to regulate synaptic transmission, learning and memory, and other fundamental processes. One of the legendary effects of cannabinoid drugs is increased motivation to eat, or hyperphagia. The two main human endocannabinoids are anandamide and 2-AG, both of which are present in C. elegans¹.

The NemaMetrix ScreenChip System is a microfluidics platform for recording electropharyngeograms (EPGs) from the nematode worm Caenorhabditis elegans and other nematode species. A key feature of the system is semi-automated loading from a reservoir of hundreds of nematodes, which increases throughput by 10 to 100 fold over previous methods for counting pharyngeal pumps. This technical note demonstrates the utility of the ScreenChip system in pharmacology, taking as an example compounds chemically related to Δ⁹-tetrahydrocannabinol (THC), the primary psychoactive compound in marijuana (Cannabis sativa). Here the ScreenChip is used to test the hypothesis that anandamide produces hyperphagia in C. elegans, a potential correlate of marijuana munchies.

Results

Well-fed adult worms were pre-exposed to the drug by soaking them for 20 min in a tube of fluid containing 100 μM anandamide. Control worms received the same treatment but without the drug. Immediately before recording, a suspension of edible bacteria was added to the tube. Worms were drawn from this tube into the chip and recorded individually for 3 min. Pumping was activated in both groups of worms but anandamide approximately doubled the pumping frequency (Fig. 1a). The overall decline in pumping frequency with time possibly resulted from depletion of food in the vicinity of the worm’s mouth during the recording (Fig. 1b).

Conclusion

We conclude that anandamide causes hyperphagia in C. elegans. This finding provides the basis for genetic screens to identify cannabinoid signaling pathways.

Methods

Strains, cultivation, and food
Synchronized worms (N2) were cultivated at 20^oC to the first day of adulthood on plates containing nematode growth medium (NGM) seeded with E. coli OP50^2,3. During EPG recordings, worms were fed Comamonas sp. at an initial concentration of 0.8 optical density units, suspended in M9 buffer³.

Electropharyngeograms
Pharyngeal pumping frequency was measured using methods described in the ScreenChip Quick Start User Guide. In the experimental group, the food suspension contained 100 μM anandamide.

References

1. Lucanic, M., Held, J. M., Vantipalli, M. C., Klang, I. M., et al. N-acylethanolamine signaling mediates the effect of diet on lifespan in Caenorhabditis elegans. Nature 473, 226-229 (2011).
2. Stiernagle, T. Maintenance of C. elegans. (2006).
3. Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A. & Cerón, J. Basic Caenorhabditis elegans methods: synchronization and observation. JoVE (Journal of Visualized Experiments) e4019-e4019 (2012).

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Background

Humans have over 70 potassium channel genes, but only some of these have been linked to disease. In this respect, the KCNQ family of potassium channels is exceptional: mutations in four out of five KCNQ genes underlie diseases including cardiac arrhythmias, deafness, and epilepsy. For example, mutations in KCNQ1 are associated with heart arrhythmias such as long-QT syndrome (LQTS), in which the cardiac action potential is prolonged. Mutations in KCNQ2 and KCNQ3 are associated with benign neonatal epilepsy (1, 2).

Homologs of KCNQ genes are found in a wide range of model organisms, where they often have functions analogous to those in humans (see Appendix). Mice bearing mutations in the mouse gene Kcnq1 exhibit epileptic seizures, coupled with aberrant cardiac events such as long pauses (3). In the Drosophila heart, a null mutation in the fly gene KCNQ, which is homologous to the human gene KCNQ1, mimics both LQTS and the effect of aging on the heart, resulting in longer cardiac contractions, increased inter-pump duration, and lower heart rate (4).

Purpose

In this technical note, we investigated the effect of mutations of orthologous KCNQ-like genes in the nematode C. elegans on electrical excitability. kqt-1 is orthologous to the subfamily of human KCNQ genes that comprise KCNQ2 to KCNQ5, and is mainly expressed in the muscles of the pharynx, a rhythmic muscular pump involved in feeding (5). kqt-2, which is  only expressed in intestinal cells, does not possess a human orthologue (5). kqt-3 is orthologous to the human gene KCNQ1 and although it is not expressed in the pharynx, kqt-3 is present in mechanosensory and chemosensory neurons, which can regulate feeding behavior (5). We hypothesized that mutations in kqt-1 and kqt-3 induce abnormalities in pharyngeal pumping that are reminiscent of cardiac arrhythmias. Taking advantage of the NemaMetrix ScreenChip system, we tested this hypothesis by measuring the duration of pharyngeal action potentials and related parameters of pumping behavior.

Methods

Strains and cultivation
The null mutant strains kqt-1(aw3), kqt-3a(aw1) and kqt-3b(aw4) were kindly donated by Dr. Aguan Wei (6). kqt-1(aw3) mutants have a 620 bp deletion of the 2.4 Kb long kqt-1 gene. kqt-3a(aw1) and kqt-3b(aw4) mutants have deletions of 1674 bp and 596 bp of the 3.1 Kb long kqt-3 gene, respectively. Synchronized worms were cultivated at 20 oC until the first day of adulthood on plates containing nematode growth medium (NGM) seeded with E. coli (OP50).

Recording electropharyngeograms (EPGs) and data analysis
Worms were pre-incubated for 20-30 min in M9 buffer containing 10 mM serotonin to induce pumping. EPGs were recorded using the NemaMetrix ScreenChip System, following methods described in the ScreenChip User Guide (7). Each recording was 2 to 3 min in duration. Recordings were analyzed using NemAnalysis software which automatically identifies individual pumps and extracts mean pump duration, mean inter-pump interval (IPI) duration and the mean pump frequency for each worm.

Results

In kqt-1(aw3) mutants, the absence of functional kqt-1 in pharyngeal muscles was associated with a significant increase in the duration of pharyngeal action potentials (35%; panel A) and length of inter-pump intervals (41%; panel B), together with a reduction in pump frequency (23%; panel C). Also, these mutants occasionally exhibited periods in which pumping appeared to cease altogether for up to three seconds. The absence of functional kqt-3, in the alleles kqt-3a(aw1) and kqt-3b(aw4), was also associated with prolonged action potential duration (Panel A), providing evidence for indirect effects on pharyngeal activity.

Discussion

We present evidence that two C. elegans genes, kqt-1 and kqt-3 are necessary for normal pharyngeal pumping, consistent with the well-known role of KCNQ potassium channel mutations in generating cardiac arrhythmias in humans and model organisms (see Appendix). Both genes are required for normal pump frequency as elicited by serotonin, whereas kqt-1 is also required for normal inter-pump intervals. We propose that the strains tested here, and the recording methodology used, could be the basis of future screens to identify pharmacological agents to mitigate certain arrhythmias.

Taken together, these data demonstrate the feasibility of using C. elegans to identify candidate genes for heart disease and to assess the effects of new therapeutic agents in high-volume, whole-animal screens in an unbiased manner.

Future Directions

The present study focused on using C. elegans as a model for cardiac arrhythmias (10), but C. elegans can also be used as a model for other KCNQ-related phenotypes, including epilepsy (8,9). C. elegans epilepsy models exhibit localized or whole-body contractions which can silence pharyngeal pumping (8,9,11). The ScreenChip system makes pharyngeal pumping easier to quantify than body contraction, providing a novel means of screening for compounds that might prevent epileptic convulsions.

References

1. Singh et. al, Nat. Genet. (1998).
2. Singh et al., Brain 126, 2726–37 (2003).
3. Goldman, A. M. et al. , Sci. Transl. Med. 1, 2ra6 (2009).
4. Ocorr, K. et al., Proc. Natl. Acad. Sci. U. S. A. 104, 3943–8 (2007).
5. Wei et al., J. Biol. Chem. 280, 21337–21345 (2005).
6. Wei et al. Nucleic Acids Res. 30, e110 (2002).
7. NemaMetrix ScreenChip System User Guide – nemametrix.com
8. Williams et al., Hum. Mol. Genet. 13, 2043–59 (2004).
9. Petersen et al., PNAS (2004).
10. Schüler et al., Sci. Rep. (2015).
11. Pandey et al., Seizure (2010).

Appendix

Preliminary Findings

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• Strain CL4176 accumulates amyloid-β1-42 in body wall muscles after shifting from 15o to 25 oC during L4 (Link et al. 2013).
• 48 h after the upshift, control strain CL802 showed normal EPG activity whereas CL4176 showed an ~25% decrease in pump frequency (A and data not shown), resulting from the increased probability of long inter-pump intervals (B).