Kathleen Conery

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Purpose

The neuromodulator serotonin (5-hydroxytryptamine; 5HT) is often used to stimulate pharyngeal pumping in C. elegans and other nematodes (e.g., 1,2,3). To investigate the concentration-dependence of 5HT treatment on pump frequency measured in the ScreenChip platform, we tested the effects of 0, 2, 5 and 10 mM 5HT on N2 (wild type) adults on Day 1. Worms were pre-incubated in 5HT for 20 min to reach steady-state pump frequency, followed by recording electropharyngeograms (EPGs) from individual worms using the ScreenChip platform (see Methods).

 

Results

The table and histogram below present pump frequency for the four experimental groups, with a representative EPG recording from the 10 mM group below.

5HT Concentration vs. Pump Frequency in C. elegans - bar graph

Table showing mean epg pump frequency from C. elegans after exposure to different concentrations of 5HT

Mean pump frequency was <0.5 Hz in the absence of 5HT and exceeded 5 Hz in 10 mM 5HT. There was a statistically significant difference between groups as determined by one-way ANOVA (F(3,127) = 95.13, p = 2.5E-32). Post hoc tests showed that all means differed significantly (p < 0.01) except for the 5 mM and 10 mM 5HT groups (Tukey’s HSD test).

5HT Concentration vs. Pump Frequency in C. elegans - bar graph

Conclusions

As expected, serotonin (5HT) stimulated pharyngeal pumping in C. elegans N2 adults in the ScreenChip system, in a concentration-dependent manner. Depending on 5HT concentration, pump frequency varied by an order of magnitude, from ~ 0.5 Hz to ~ 5 Hz. Saturation of the effect was reached by 5 or 10 mM 5HT. In a previous study, 10 mM 5HT was used to elicit sustained pharyngeal pumping in C. elegans, against which the effects of inhibitory drugs were tested in 8-channel microfluidic EPG chips (3). We recommend the same testing protocol for the ScreenChip system.

 

Methods

C. elegans synchronization and cultivation
Synchronized N2 C. elegans were obtained via a bleach synchronization procedure. The resulting L1s were cultivated on plates containing standard nematode growth medium (NGM) seeded with E. coli OP50 and allowed to grow at 20 ºC until they reached the first day of adulthood (Day 1) (4,5).

Solutions
The 10 mM 5HT solution was made in a 15 mL conical vial by adding 10 mL of M9 buffer to 43 mg 5HT. This stock was diluted 2:1 and 5:1 with M9 to obtain lower concentrations. All 5HT was used within 2 h of being prepared.

Recording electropharyngeograms (EPGs)
A glass pipette with M9 buffer was used to rinse Day1 adults from a plate. They were transferred to a 1.5 mL Eppendorf tube and centrifuged for 2 min at 6,000 RPM. The supernatant was removed and replaced with fresh M9 and the worms were spun down again. The supernatant was removed again and replaced with M9 containing the desired 5HT concentration. Worms were incubated for 20 min to activate pharyngeal pumping. Pump frequency was measured from EPG recordings using the NemaMetrix ScreenChip 100 system, as described in the ScreenChip User Guide (6). Each EPG recording was 2 to 4 min in duration and the experiment was replicated in triplicate on different days.

 

References

  1.  Raizen, D.M., Avery, L., 1994. Electrical activity and behavior in the pharynx of Caenorhabditis elegans. Neuron 12, 483–4951
  2.  Tahseen, Q., Sheridan, J., Perry, R.N., 2003. Electropharyngeograms of pharyngeal pumping activity in six species of free-living nematodes. Nematology 6, 49–54
  3.  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
  4.  Porta-de-la-Riva, M et al. 2012. Basic Caenorhabditis elegans methods: synchronization and observation J Vis Exp 64:4019
  5.  Stiernagle T. 2006. Maintenance of C. elegans. WormBook, http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
  6.  NemaMetrix ScreenChip User Guide. www.nemametrix.com

 

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