Behavioural assay for olfactory plasticity in C. elegans

• Protocol
• Discussion

Authors: Takaaki Hirotsu, Yu Hayashi, Ryo Iwata, Hirofumi Kunitomo, Eriko Kage-Nakadai, Takeo Kubo, Takeshi Ishihara & Yuichi Iino

Introduction

C. elegans shows odour adaptation after continuous exposure to an odorant for more than 30 minutes (1). We recently found that odour adaptation is also observed after a short pre-exposure (5 min) to odorants (2). This new type of adaptation requires several interneurons including AIY and RIF, indicating that this behavioural plasticity depends on neural circuits.

The protocol for the short pre-exposure adaptation was modified from the conventional adaptation assay (1). It is important that the plate format used in this protocol (Fig 1a) is different from that used in the conventional adaptation assays (Fig 1b). In our format, spots of odorants, worms and control spots are aligned in close proximity. Therefore, our format is more sensitive to odour-avoidance behaviour after odorant pre-exposure.

Reagents

Wash buffer

• 5 mM potassium phosphate (pH 6.0)
• 1 mM CaCl2
• 1 mM MgSO4
• 0.5 g/l gelatin

Assay plates (9cm plates)

• 5 mM potassium phosphate (pH 6.0)
• 1 mM CaCl2
• 1 mM MgSO4
• 2 % Bacto agar (Difco)

Odorants

• isoamyl alcohol, benzaldehyde, diacetyl, pyrazine

• Odorants spotted on assay plates are diluted in EtOH. Odorants used in the pre-exposure steps are diluted in water.

• 1 M sodium azide

Equipment

• Microfuge

Procedure

1. 4 days before the assay, pick about 6 adults (depending on the brood size of strains; 6 adults are suitable for WT) to NGM plates, where the bacterial strain NA22 is seeded.
2. Collect well-fed animals in microfuge tubes and wash 3 times with Wash buffer to remove bacteria and larval worms. After the last wash, remove as much of the supernatant as possible.
3. Add 100 μl of 10e-4 dilutions of odorants in water into the tubes for the odorant pre-exposure.
4. During the odorant pre-exposure, prepare assay plates with the format drawn in Fig. 1a. Spot 1 μl each of odorant and 0.5 μl each of 1 M sodium azide on two points at one end of the plates, and spot only sodium azide on the other side.
5. After 5 min of pre-exposure, add 1 ml of Wash buffer and centrifuge for 5 seconds at 100 g.
6. Suck animals settled at the bottom and spot about 50 animals at the center of an assay plate. The number of animals spotted on a plate is critical, because when a greater number of animals are spotted, they tend to form clumps and fail to leave the origin.
7. Remove excess liquid with Kimwipes, and at the same time disperse the animals gently along the midline of the plates, so that they do not form clumps.
8. Incubate for 30 min at 23 ± 1 ºC. Keeping the temperature in this range is critical, because the chemtaxis behaviour is strongly influenced by temperature.
9. After 30 min, count the number of animals in areas A or B (Fig. 1a), while animals that remain within 0.5 cm of the midline are not counted to exclude immotile animals from consideration. Calculate the chemotaxis index as (NA – NB) / (NA + NB).

References

1. Colbert, H. A. & Bargmann, C. I. Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans. Neuron 14, 803-812 (1995)
2. Hirotsu, T., and Iino, Y. Neural circuit-dependent odor adaptation in C. elegans is regulated by the Ras-MAPK pathway. Genes Cells 10, 517-530 (2005)

Acknowledgements

We thank K. Yamada for useful advice and discussion.

Figures

Fig. 1: Layout of assay plates used for olfactory plasticity assay.

Associated Publications

A trophic role for Wnt-Ror kinase signaling during developmental pruning in Caenorhabditis elegans, Yu Hayashi, Takaaki Hirotsu, Ryo Iwata, Eriko Kage-Nakadai, Hirofumi Kunitomo, Takeshi Ishihara, Yuichi Iino, and Takeo Kubo, Nature Neuroscience 12 (8) 981 - 987 28/06/2009 doi:10.1038/nn.2347

Author information

Takaaki Hirotsu, Department of Biology, Graduate School of Science, Kyushu University/ Deparment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo

Yu Hayashi, Department of Biological Sciences, Graduate School of Science, The University of Tokyo/ Present address: Laboratory for Behavioral Genetics, RIKEN Brain Science Institute

Ryo Iwata, Hirofumi Kunitomo & Yuichi Iino, Deparment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo

Eriko Kage-Nakadai, Department of Biological Sciences, Graduate School of Science, The University of Tokyo/ Present address: Department of Physiology, Tokyo Women's Medical University, School of Medicine

Takeo Kubo, Department of Biological Sciences, Graduate School of Science, The University of Tokyo

Takeshi Ishihara, Department of Biology, Graduate School of Science, Kyushu University

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.139. Originally published online 3 July 2009.