Shawn Lockery, Ph.D.

NemaMetrix logo - C. elegans research and phenomics


Purpose

In C. elegans, a key marker of age-related decline in health is a reduction in the rate of pharyngeal pumping. Until now, it has been necessary to count pumps by eye, which is laborious, slow, and often inaccurate. The NemaMetrix ScreenChip System automates the process of counting pumps by recording their electrical signature, called the electropharyngeogram (EPG). Here we show that the age-related decline in pumping is recapitulated when pumps are counted in electrical recordings.

Fig. 1. Pump frequency decreases with age. Mean pump frequency of the C. elegans reference strain (N2) in buffer containing 10 mM serotonin; error bars are standard error of the mean. Statistics: linear regression slope is different from zero at p < 10-4.

Fig. 1. Pump frequency decreases with age. Mean pump frequency of the C. elegans reference strain (N2) in buffer containing 10 mM serotonin; error bars are standard error of the mean. Statistics: linear regression slope is different from zero at p < 10-4.

Methods

Populations of N2 worms were synchronized via a bleaching procedure and grown to larval stage L4 on agarose plates containing Nematode Growth Medium (NGM). L4 animals were transferred to plates containing NGM + 50 μM 2′-Deoxy-5-fluorouridine(FUDR) to prevent egg laying. Worms were transferred to fresh NGM + FUDR plates every ~3 days to ensure a plentiful supply of food and to limit contamination (1,2). On the appropriate day of testing, worms were washed in M9 4-5 times, then exposed to 10 mM serotonin in M9 for 20 minutes prior to recording. 15-30 animals were recorded at each stage of adulthood for 2 minutes (3). Individual pumps were identified automatically using NemAnalysis software and pump frequency was computed as the number of pumps divided by total recording time.

Comparative EPG recordings of C. elegans at different ages.

Fig. 2. Typical electrophysiological waveforms and morphologies on Day 1 and Day 13 from the investigation in Fig. 1. In the photographs, the dark object is the worm and the lighter object is a worm-like feature in the ScreenChip, which is 1 mm long; anterior it to the right. In the electrical traces, voltage is shown in arbitrary units.

Choosing Which Chip Size to Use

The Medium ScreenChip (40 micrometer channel width) is suitable for Day 1- Day 4 adults (C. elegans). Beginning on Day 5, the Large ScreenChip (60 micrometer channel width) is required, which can be used up to Day 15. However, Days 4-6 can be inconsistent in terms of optimal chip size. On these days it is helpful to test the goodness of fit before each recording session.

General Troubleshooting for the ScreenChip System and C. elegans age

References

  1. Caenorhabditis Intervention Testing Program Standard Operating Procedure: Manual Lifespan Assay Plate Preparation. 2015.
  2. Sutphin G.L., Kaeberlein M. 2009, Measuring Caenorhabditis elegans Life Span on Solid Media, Journal of Visualized Experiments,
  3. ScreenChip System Quick Start User Guide, http://nemametrix.com/downloads/