Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Aug:16:174-187.
doi: 10.1016/j.ijpddr.2021.05.005. Epub 2021 Jul 2.

Comparison of electrophysiological and motility assays to study anthelmintic effects in Caenorhabditis elegans

Steffen R Hahnel  1 , William M Roberts  2 , Iring Heisler  3 , Daniel Kulke  4 , Janis C Weeks  5
Affiliations

Comparison of electrophysiological and motility assays to study anthelmintic effects in Caenorhabditis elegans

Steffen R Hahnel et al. Int J Parasitol Drugs Drug Resist. 2021 Aug.

Abstract

Currently, only a few chemical drug classes are available to control the global burden of nematode infections in humans and animals. Most of these drugs exert their anthelmintic activity by interacting with proteins such as ion channels, and the nematode neuromuscular system remains a promising target for novel intervention strategies. Many commonly-used phenotypic readouts such as motility provide only indirect insight into neuromuscular function and the site(s) of action of chemical compounds. Electrophysiological recordings provide more specific information but are typically technically challenging and lack high throughput for drug discovery. Because drug discovery relies strongly on the evaluation and ranking of drug candidates, including closely related chemical derivatives, precise assays and assay combinations are needed for capturing and distinguishing subtle drug effects. Past studies show that nematode motility and pharyngeal pumping (feeding) are inhibited by most anthelmintic drugs. Here we compare two microfluidic devices ("chips") that record electrophysiological signals from the nematode pharynx (electropharyngeograms; EPGs) ─ the ScreenChip™ and the 8-channel EPG platform ─ to evaluate their respective utility for anthelmintic research. We additionally compared EPG data with whole-worm motility measurements obtained with the wMicroTracker instrument. As references, we used three macrocyclic lactones (ivermectin, moxidectin, and milbemycin oxime), and levamisole, which act on different ion channels. Drug potencies (IC50 and IC95 values) from concentration-response curves, and the time-course of drug effects, were compared across platforms and across drugs. Drug effects on pump timing and EPG waveforms were also investigated. These experiments confirmed drug-class specific effects of the tested anthelmintics and illustrated the relative strengths and limitations of the different assays for anthelmintic research.

Keywords: 8-channel chip; Anthelmintics; Caenorhabditis elegans; Electropharyngeogram (EPG); Levamisole; Macrocyclic lactones; Nematode pharynx; Pharyngeal pumping; ScreenChip; wMicroTracker.

PubMed Disclaimer

Conflict of interest statement

Iring Heisler is an employee of Elanco Animal Health and Daniel Kulke, and Steffen R. Hahnel were employees of Elanco Animal Health at the time the work was undertaken. Elanco Animal Health develops and sells veterinary pharmaceuticals including dewormers. Janis C. Weeks and William M. Roberts hold equity in InVivo Biosystems Inc., a company that develops and sells laboratory devices such as microfluidic EPG chip platforms and the wMicroTracker reported here. Except for the authors, Elanco Animal Health and InVivo Biosystems Inc. were not involved in the preparation of the manuscript. The decision to publish the manuscript was jointly taken.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Experimental setup of microfluidic electropharyngeogram (EPG) recording platforms and wMicroTracker motility assay. A. ScreenChip system. i. The microfluidic chip has an inlet port at one end and an outlet port at the other. Suction applied to the outlet port is used to load worms into a collecting chamber (not shown), where they remain until individual worms are moved into a microchannel (inset) and positioned between two indium tin oxide (ITO) electrodes (represented by E1, E2; the electrodes are off screen) for recording. ii. Representation of EPGs recorded serially in the ScreenChip. Gaps (dotted lines) indicate when worms are being positioned between recordings. iii. Experimental protocol. At t = 0, drug or solvent was added to worms suspended in M9 buffer containing 10 mM 5HT (“M9-5HT”) and incubated for 30 min. Thirty-second recordings from each worm were made between t = 30 and t = 60 min (orange shading). B. 8-channel EPG platform. i. The chip has a branching network of microchannels (filled with red dye in this image) that distribute worms into 8 recording modules (labelled 1 to 8). Each recording module has an associated recording electrode (blue wire). After loading worms through the input port, the loading tubing is removed and a hollow common reference electrode (not shown) is inserted into the input port, through which solutions are perfused. Reproduced from Lockery et al. (2012) with permission from the Royal Society of Chemistry. ii. The 8-channel chip records EPGs simultaneously from 8 worms, here shown in M9-5HT. iii Experimental protocol. EPGs were recorded for 75 min. During the first 15 min, M9-5HT was perfused to obtain baseline activity (green shading). At t = 0 min, the perfusate was switched to M9-5HT containing either drug or solvent control, and recordings were continued for 60 min more (orange shading). C. wMicroTracker. i. The wMicroTracker is a multi-well plate reader that quantifies worm motility in liquid media by counting the number of times an infrared LED microbeam is interrupted by worms moving in a well. ii. Experimental protocol. Worms suspended in M9 containing 0.001% Triton-X were aliquoted into the 96 wells of a plate (~70 worms/well). At time t = 0, drug or solvent was pipetted into each well to obtain the desired concentration. Motility was measured continuously for 4 h (orange shading) and analyzed in 30 min bins. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
Effects of MLs and LEV on pump frequency in the ScreenChip. A Representative EPGs recorded from worms in ScreenChips after treatment with two different IVM concentrations for >30 min. Each pump waveform (example marked by dotted box) is demarcated by an E (excitation) spike and R (relaxation) spike (Raizen and Avery, 1994). By convention, the E spike is oriented upward. B. Concentration-dependent inhibition of pharyngeal pumping. Each drug was tested at five concentrations, with mean pump frequencies (Hz) calculated from 15 worms in each of three to four independent experiments per group. The panel shows plots (mean ± S.E.M.) of normalized mean pump frequencies. Hill curves were fit to the normalized data using a maximum likelihood criterion with three free parameters (see Methods). A dashed horizontal line at y = 0.5 intercepts the IC50 value for each fitted curve; the IC50 values appear in Table 1. The three MLs (IVM, MOX, MIL) and LEV all caused a concentration-dependent decrease in pump frequency. MOX and IVM were the most potent, followed by MIL and LEV. Statistical comparisons between IC50 values appear in Table 1. Hill slope coefficients: LEV, 2.3; MIL, 2.1; IVM, 2.4; MOX, 3.8.
Fig. 3
Fig. 3
Time course of ML and LEV effects on pump frequency in the 8-channel platform. A-C each show a set of representative EPG recordings from worms within a single chip. Each worm is numbered. At the compressed time scale shown, changes in amplitude are sometimes apparent, but individual pumps cannot always be resolved. Baseline activity in M9-5HT was recorded for 15 min, followed by switching the perfusate (vertical grey bar) to M9-5HT containing the indicated solution. A. Pumping continued steadily when switched from M9-5HT to M9-5HT. B. In 10 μM MOX, all worms quit pumping by ~25 min. C. In 3.3 mM LEV, pumping frequency transiently decreased, showed partial recovery and then, in most worms shown, decreased again. D-G are plots of pump frequency [mean (line) ± S.E.M. (shading)] over time in different concentrations of the drugs as indicated, with the number of worms in each treatment group in parentheses. In each plot, the final 5 min of baseline pumping is shown and the electrical artifact from switching the perfusate is blanked. A vertical dotted line indicates t = 0 when the new solution reached the worms. The DMSO concentration in all groups was 0.1%. The MLs (D–F) all caused a smooth, concentration-dependent decrease in pump frequency over time. In contrast, LEV (G) showed transient and sustained phases of inhibition (Weeks et al., 2018b).
Fig. 4
Fig. 4
Effects of MLs and LEV on pump frequency in the 8-channel platform. The panel shows plots (mean ± S.E.M.) of normalized mean pump frequencies during a 5 min epoch from t = 55–60 min plotted against drug concentration for the three MLs and LEV (color legend in the key). Pump frequencies were normalized to each worm's baseline pump frequency before averaging across worms. Hill curves were fit to the normalized data using a maximum likelihood criterion with three free parameters (see Methods); the dashed horizontal line at 0.5 intercepts the IC50 value for each drug. IC50 values and statistical comparisons are in Table 1. Hill slope coefficients: LEV, 2.4; MIL, 2.6; IVM, 1.4; MOX, 0.90. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5
Fig. 5
Effects of MLs and LEV on the temporal pattern of pumping in the 8-channel platform. Inter-pump intervals (IPIs) were measured as the time between successive E spikes. A - D, IPI values during the epoch t = 0–60 min were plotted as probability density functions for three MLs and LEV (same data set as Fig. 3). Keys show color coding of drug concentrations. The mode (most probable value of IPI) is indicated by an arrow in A. In IVM, MOX and MIL (A–C), the mode was maintained at all concentrations tested while the probability of longer IPIs increased with drug concentration. Similarly, LEV (D) caused a concentration-dependent increase in the probability of longer IPIs but, unlike the MLs, additionally caused a rightward shift of the mode to longer IPIs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6
Fig. 6
Effects of MLs and LEV on EPG waveforms in the 8-channel platform. For each drug, worms were analyzed in 3 chips, with n = 19 to 22 worms per drug. We analyzed EPG waveforms of worms that ceased pumping after the perfusate switch; in LEV, the worms were in the sustained phase of inhibition following the switch (see Fig. 3G). A. Representative EPG waveforms in three MLs during the baseline period (“Before”) and in the presence of the indicated ML, as pumping neared its end in individual worms (“After”; in 10 μM IVM, 1 μM MOX and 1 μM MIL). For each drug, Before and After traces are shown for the same worm. In all three MLs, EPG waveforms become smaller in amplitude and briefer. B. Representative changes in EPG waveforms in LEV. The traces are from seven worms in one chip, during the baseline period (left) and 7.5 min after switching to 10 mM LEV (right). E and R spikes persisted but a conspicuous “hump” appeared after R spikes (red arrows). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7
Fig. 7
Time course of ML and LEV effects on motility in the wMicroTracker. A – D are plots of normalized activity (mean ± S.E.M.; see Methods) over time in different concentrations of the drugs indicated. Activity was recorded continuously for 4 h and binned into 30 min epochs for analysis. Points in the plots are centered in the middle of each time bin. The MLs (A–C) and LEV (D) all caused a concentration-dependent decrease in motility over time.
Fig. 8
Fig. 8
Comparison of concentration-response curves from different assays. Each panel shows concentration-response curves for one drug, using the three assays indicated. Key in A applies to all panels. ScreenChip and 8-channel curves are repeated from Fig. 2, Fig. 4, respectively, while wMicroTracker curves were plotted from the final time bin (t = 210–240 min) shown in Fig. 7. IC50 values and statistics for wMicroTracker data are shown in Table 1. Hill slope coefficients for wMicroTracker data were: IVM, 2.7; MOX, 2.6; MIL, 3.8; LEV, 2.4.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ardelli B.F., Stitt L.E., Tompkins J.B., Prichard R.K. A comparison of the effects of ivermectin and moxidectin on the nematode Caenorhabditis elegans. Vet. Parasitol. 2009;165:96–108. - PubMed
    1. Auner A.W., Tasneem K.M., Markov D.A., McCawley L.J., Hutson M.S. Chemical-PDMS binding kinetics and implications for bioavailability in microfluidic devices. Lab Chip. 2019;19:864–874. - PMC - PubMed
    1. Avery L., Horvitz H.R. Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans. J. Exp. Zool. 1990;253:263–270. - PubMed
    1. Avery L., You Y.J. C. elegans feeding. WormBook. 2012:1–23. - PMC - PubMed
    1. Blanchard A., Guegnard F., Charvet C., Crisford A., Courtot E., Sauvé C., Harmache A., Duguet T., O'Connor V., Castagnone-Sereno P., Reaves B., Wolstenholme A.J., Beech R.N., Holden-Dye L., Neveu C. Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: when novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species. PLoS Pathog. 2018;14 - PMC - PubMed