Pentylenetetrazole (PTZ)-induced Convulsion Assay to Determine GABAergic Defects in Caenorhabditis elegans

Materials and Reagents

1. 35 mm and 60 mm Petri dishes (35 mm: Tarsons, catalog number: 460035 ; 60 mm: Tarsons, catalog number: 460061 )
2. Spreader (Tarsons, catalog number: 920081 )
3. 99.99% platinum wire (Sigma-Aldrich, catalog number: 267201 )
4. C. elegans: N2 (Wild type worms)(University of Minnesota, Caenorhabditis Genetic Center) and casy-1(tm718) (National Bio resource Project, Japan)
5. Escherichia coli OP50 (University of Minnesota, Caenorhabditis Genetic Center)
6. Pentylenetetrazole (PTZ) (Sigma-Aldrich, catalog number: P6500 )
7. Ethanol (Ethyl alcohol Absolute, ACS-ISO grade) (Merck, catalog number: 100983 )
8. Cholesterol (SRL Sisco Research Laboratories, catalog number: 54181 )
9. Calcium chloride dihydrate (CaCl₂•2H₂O) (Sigma-Aldrich, catalog number: C3306 )
   
10. Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M7506 )
11. Potassium phosphate, monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5379 )
12. Potassium phosphate, dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: P8281 )
13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
14. Bacto-agar (HiMedia Laboratories, catalog number: GRM026 )
15. Bacto-peptone (SRL Sisco Research Laboratories, Bacto^TM, catalog number: 95292 )
16. 100 mg/ml PTZ stock solution (see Recipes)
17. 5 mg/ml cholesterol (see Recipes)
18. 1 M CaCl₂ stock solution (see Recipes)
19. 1 M MgSO₄ stock solution (see Recipes)
20. 1 M KPO₄, pH 6.0 stock solution (see Recipes)
21. Nematode growth medium (NGM) agar plates (see Recipes)
    

Equipment

1. Pipettes (Eppendorf, model: Research^® plus, catalog number: 2231000224 )
2. 2 L glass conical flask (DWK Life Sciences, DURAN, catalog number: 2121763 ) 
3. Autoclave (Equitron-7431 SLEFA)
4. Microscope (ZEISS, model: Stemi 2000 C )
   

Software

1. GraphPad Prism v6 (GraphPad Software)

Procedure

1. For routine growth and maintenance of C. elegans, see Wormbook chapter Maintenance of C. elegans (Stiernagle, 2006). Maintain C. elegans strains to be used in the assay in plates without contamination.
2. Prepare fresh 35 mm Nematode Growth Medium (NGM) plates (see Recipes) containing 3 ml media, at least one day before the assay and store at 4 °C until use.
3. Seed regular 60 mm NGM agar plates for routine maintenance with 15 μl E. coli OP50 under sterile conditions in a bacterial hood two days before the assay. Allow the plates to dry at room temperature.
4. Place about 20-30 L4 stage wild-type (WT), N2, and mutant strains of interest using a pick made of platinum wire (described in Stiernagle, 2006) onto the 60 mm NGM plates seeded with E. coli OP50 and allow the worms to grow at 20 °C for about 20 h.
5. On the day of assay, take out 35 mm NGM plates from 4 °C and allow them to attain room temperature. Pipette 300 μl of 100 mg/ml PTZ stock solution at the center of the plate and spread it uniformly over the entire NGM surface to obtain a final concentration of 10 mg/ml. Allow the plates to dry for 35 min with lids closed. Keep the same number of 35 mm NGM plates without PTZ to be used as control in the experiment. Spread 300 μl of Ethanol on control plates to avoid behavioral changes due to exposure of ethanol. Once the plates are dried, put a 10 μl drop of concentrated E. coli OP50 (overnight culture concentrated by centrifugation) at the center of the plate and let it dry for at least 2 h at room temperature. The plates are now ready for the assay.
6. Transfer 10-15 worms of each genotype from Step 4 onto the center of the plate on food on both the control and PTZ-containing plates. Leave the plates undisturbed for 30 min at room temperature (~23 °C).
7. Observe the C. elegans after the 30 min interval. Count the total number of animals on the plate and the number of C. elegans showing anterior convulsions or ‘head bobs’ (Figure 2A). Wild type C. elegans shows no response to PTZ exposure (S1 movie in Thapliyal et al., 2018). While mutant defective in GABAergic synaptic transmission show high degree of convulsions (S2 movie in Thapliyal et al., 2018). One can also observe the C. elegans after a 60 min interval to take the second reading (optional). These recordings are also done at room temperature (~23 °C).
   
   
   Figure 2. casy-1 mutants show increased convulsions when compared with WT C. elegans. The casy-1 mutants show higher sensitivity to the GABA receptor antagonist PTZ when compared to WT animals. A. Representative still frame images demonstrating casy-1 mutant C. elegans with anterior convulsions (head bobs) and tail shrinker phenotype. The still frame images are representative frames from movies (7 frames/sec). B. The graph shows the fraction of animals showing anterior ‘head bobs’ after 30 min and 60 min exposure to 10 mg/ml PTZ. The sensitivity to PTZ could be fully rescued by expressing casy-1 isoforms in GABAergic neurons but not in cholinergic neurons or muscle. Assays were done (~15 C. elegans/assay) at least thrice. Values that differ significantly from WT animals are indicated (*** indicates P < 0.0001 using two-way ANOVA). Data are represented as mean ± S.E.M. Part of Figure 2A and Figure 2B have been previously published in (Thapliyal et al., 2018).
   
   
8. Here in the present example, casy-1 represents a cell adhesion molecule that has been reported to regulate vesicle trafficking in GABAergic motor neurons (Thapliyal et al., 2018). GABA release from the presynaptic terminals is reduced in casy-1 mutants which can be easily interpreted from high degree of convulsions in the mutant upon PTZ exposure.
   

Data analysis

1. Calculate the percentage of C. elegans showing convulsions on each plate at the 30 and 60 min time point using the formula given below:
   
   
   
   
2. Software: GraphPad Prism v6.
3. Use Grouped graph with 3 replicate values to plot the results. At least 10-15 worms of each genotype are used per assay. Repeat the assay two more times on different days (total 3 trials) and report the summary of the statistics as shown in Figure 2B.
4. Statistical analysis: A two way-ANOVA is used to determine statistical P-values.
   

Notes

1. As a standard scientific procedure to avoid biases, the experimenter should be blinded to the genotypes of the test C. elegans. Ideally, a colleague from the lab can number the plates to prevent the experimenter from identifying the genotypes. For mutants with visual phenotypic defects, an additional mutant line with a similar phenotypic defect and unrelated to the content of the study must be added.
2. The sensitivity of the C. elegans to PTZ could change depending upon the variability in experimental conditions (room temperature, humidity etc.) or differences in the PTZ stock obtained from the company. However, the overall readout of the experiment usually remains the same.
3. In our hands, more variability in convulsions was seen in C. elegans containing extrachromosomal arrays (rescue lines). This could be due to differences in array lines and should not be concluded as a generalized phenomenon.
   

Recipes

1. 100 mg/ml PTZ Stock Solution
   Dissolve 100 mg PTZ in 1 ml volume of absolute ethanol
   This solution is prepared fresh before each experiment
2. 5 mg/ml cholesterol
   Add 500 mg of cholesterol in 100 ml of 95% ethanol and mix by rotating on a rotator at room temperature. It takes a few hours to dissolve
   Store at 4 °C
3. 1 M CaCl₂ stock solution
   Dissolve 14.7 g of CaCl₂•2H₂O in 100 ml ddH₂O and autoclave for 15 min at 121 °C
   Store at 4 °C
4. 1 M MgSO₄ stock solution
   Dissolve 12.04 g of MgSO₄ in 100 ml ddH₂O and autoclave for 15 min at 121 °C
   Store at 4 °C
5. 1 M KPO₄, pH 6.0 stock solution
   
   1. Mix 108.3 g KH₂PO₄ and 35.6 g K₂HPO₄ in 500 ml ddH₂O
   2. Adjust the pH to 6.0 by adding NaOH, finally make up the volume to 1 L
   3. Aliquot the solution and autoclave for 15 min at 121 °C
   4. Store at 4 °C
6. Nematode growth medium (NGM) agar plate
   
   1. Add 3 g NaCl, 16 g Bacto-agar, and 2.5 g Bacto-peptone in 975 ml of ddH₂O in a 2 L flask
   2. Autoclave for 50 min at 121 °C
   3. Let the NGM agar cool to 55-60 °C. Add 1 ml of 5 mg/ml cholesterol, 1 ml of 1 M CaCl₂, 1 ml of 1 M MgSO₄, and 25 ml of 1 M KPO₄, pH 6.0

Acknowledgments

This protocol has been adapted from Thapliyal et al. (2018). ST thanks the Council of Scientific and Industrial Research (CSIR) for a graduate fellowship. KB is an Intermediate Fellow of the DBT-Wellcome Trust India Alliance and thanks the Alliance for funding support (grant number IA/12/500516).

Competing interests

Authors declare no conflict of interest or competing interest.

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