Shawn Lockery, Ph.D.

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Purpose

The NemaMetrix ScreenChip System is a microfluidic platform for recording electropharyngeograms (EPGs) from 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 that the pharyngeal phenotypes of the previously identified neurotransmission mutants eat-4 (1) and avr-15 (2) are recapitulated in the ScreenChip system. Additionally, it capitalizes on increased throughput to extend these phenotypes by measuring higher-order statistics of pharyngeal pumping.

 

Results

The initiation and termination of individual pumping events were identified automatically by an off-line thresholding procedure. Despite the presence of non-classical EPG waveforms in some of mutant animals, 43 of 50 (86%) of the eat-4 recordings and 68 of 77 (88%) of the avr-15 recordings could be analyzed, which compares favorably with the fraction of usable N2 recordings, 40 of 44 (90%). This study examined pump frequency, pump duration, and inter-pump interval (Fig. 1A-C).

Data analysis of N2 and glutamate neurotransmission in C. elegans mutants eat-4 and avr-15.

Figure 1. Data analysis of N2 and glutamate neurotransmission mutants eat-4 and avr-15. A. Pump frequency. Means: N2, 5.6 Hz; eat-4, 3.9 Hz; avr-15, 5.3 Hz. B. Probability distribution of pump durations. Modal duration (i.e., peak of the duration distribution): N2, 96 ms; eat-4, 104 ms; avr-15, 88 ms. Mean duration: N2, 96 ms; eat-4, 127 ms; avr-15, 101 ms. C. Probability distribution of inter-pump intervals (IPIs) from start of one pump to start of the next. Modal IPI: N2, 165 ms; eat-4, 205 ms; avr-15, 175 ms. Mean IPI: N2, 181 ms; eat-4, 301 ms; avr-15, 194 ms. Data were recording using the ScreenChip™ System. Shading, ± SEM.

 

Data from mutants and the N2 reference strain are compared in Table 1. Consistent with previous findings in which pumps were counted by visual observation, mean pump frequency as measured by the ScreenChip System was reduced in eat-4 and avr-15. Differences in the degree of pump frequency reduction in avr-15 might be explained by differences in the way pumping was induced (Table 1, legend). Additionally, ScreenChip recordings from avr-15 reproduced the previously observed increase in mean pump duration measured by a low-throughput EPG method (3).

Comparison of pharyngeal pumping in glutamate transmission in C. elegans mutants eat-4 and avr-15, relative to N2 worms.

Table 1. Comparison of pharyngeal pumping in glutamate transmission mutants eat-4 and avr-15, relative to N2 worms. (e) EPG recording, (m) Manual counts, (s) Serotonin-induced pumping (10 mM), (f) Food-induced pumping, (u) unc-31 induced pumping, (ND) No data, (**) p < 0.03.

 

The increase in throughput afforded by the ScreenChip System facilitated the determination of probability density distributions for parameters of individual pumping events. The pump-duration distribution indicates that increased pump length in eat-4 and avr-15 is mainly the result of the prominent tails in the mutant distributions that are not seen in the reference strain. The fact that the modal pump duration was nevertheless similar in all three strains is intriguing. Bursts of inhibitory glutamatergic postsynaptic potentials, which occur during the plateau phase of pharyngeal action potentials recorded intracellularly, are believed to be a major contributor to action potential termination in C. elegans (6). A simple model that accounts for similar modal durations in mutants and N2 is that the reduction in the efficacy of action potential termination, as a result of impaired glutamate transmission, is enhanced for synaptic potentials that occur later in the burst.

The distributions of inter-pump intervals (Fig. 1C) provided a test of the hypothesis that pump frequency (the inverse of inter-pump interval), is inversely related to pump duration. Whereas pump duration was extended in both mutants (Fig. 1B), only eat-4 exhibited a substantial increase in long inter-pump intervals. Thus, an increase in pump duration does not necessarily entail a decrease in pump frequency.

 

Conclusion

This study shows that the ScreenChip System can reproduce the expected effects of pumping mutants in which glutamate transmission is disrupted. It also illustrates the advantages of increased throughput, facilitating analysis in terms of probability distributions of pumping parameters, which can provide mechanistic insights into the effects of genetic mutations and a wide range of other manipulations of nematode physiology.

 

Methods

Strains and cultivation
Synchronized worms (N2, eat-4(ky5), avr-15(ad1051)) were cultivated at 20 ºC to the first day of adulthood on plates containing nematode growth medium (NGM) seeded with E. coli(7,8).

Electropharyngeograms
Worms were washed by 5 cycles of centrifugation (2 min, 6,000 RPM) and resuspension in M9 buffer. Pumping was induced by adding serotonin (10 mM) to the buffer. In the final resuspension, the M9 contained 10 mM serotonin and the worms were incubated for 20-30 min before being recorded. Pharyngeal pumping frequency was measured using methods described in the ScreenChip User Guide (9). Each EPG recording was 2-3 min. in duration.

 

References

  1. Lee RY, Sawin ER, Chalfie M, Horvitz HR, Avery L. EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in caenorhabditis elegans. J Neurosci. 1999 Jan 1;19(1):159-67.
  2. Dent JA, Davis MW, Avery L. avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans. EMBO J. 1997 Oct 1;16(19):5867-79.
  3. Raizen DM, Avery L. Electrical activity and behavior in the pharynx of Caenorhabditis elegans. Neuron. 1994 Mar;12(3):483-95.
  4. Greer ER, Pérez CL, Van Gilst MR, Lee BH, Ashrafi K. Neural and molecular dissection of a C. elegans sensory circuit that regulates fat and feeding. Cell Metab. 2008 Aug;8(2):118-31.
  5. Steger KA, Avery L. The GAR-3 muscarinic receptor cooperates with calcium signals to regulate muscle contraction in the Caenorhabditis elegans pharynx. Genetics. 2004 Jun;167(2):633-43.
  6. Avery L, You YJ. C. elegans feeding. WormBook. 2012 May 21:1-23.
  7. Stiernagle T. Maintenance of C. elegans. WormBook. 2006 Feb 11:1-11.
  8. Porta-de-la-Riva M, Fontrodona L, Villanueva A, Cerón J. Basic Caenorhabditis elegans methods: synchronization and observation. J Vis Exp. 2012 Jun 10;(64):e4019.
  9. nemametrix.com

 

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