This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Behavioural assay for olfactory plasticity in C. elegans
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Figure 1
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.
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
Microfuge
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.
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.
Add 100 μl of 10-4 dilutions of odorants in water into the tubes for the odorant pre-exposure.
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.
After 5 min of pre-exposure, add 1 ml of Wash buffer and centrifuge for 5 seconds at 100 g.
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.
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.
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.
- 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).
- Colbert, H. A. & Bargmann, C. I. Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans. Neuron 14, 803-812 (1995)
- 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)
We thank K. Yamada for useful advice and discussion.
Arunan MC
commented on 25 July, 2009
I wonder whether I can do similar studies on the drosophila fly larvae by apropriate modifications! I will be grateful if some one tells me the status on fly larval adaptation studies. MC Arunan
Arunan MC
commented on 25 July, 2009
I wonder whether I can do similar studies on the drosophila fly larvae by apropriate modifications! I will be grateful if some one tells me the status on fly larval adaptation studies. MC Arunan
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Posted 03 Jul, 2009
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Associated Publications
A trophic role for Wnt-Ror kinase signaling during developmental pruning in Caenorhabditis elegans
by Yu Hayashi, Takaaki Hirotsu, Ryo Iwata, +4
Nature Neuroscience (10 June, 2009)
Method Article
Behavioural assay for olfactory plasticity in C. elegans
Version History
CURRENT STATUS: Posted
Version 1
Posted 03 Jul, 2009
Metrics
Comments: 2
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
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.
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
Microfuge
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.
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.
Add 100 μl of 10-4 dilutions of odorants in water into the tubes for the odorant pre-exposure.
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.
After 5 min of pre-exposure, add 1 ml of Wash buffer and centrifuge for 5 seconds at 100 g.
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.
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.
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.
- 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).
- Colbert, H. A. & Bargmann, C. I. Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans. Neuron 14, 803-812 (1995)
- 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)
We thank K. Yamada for useful advice and discussion.
Arunan MC
commented on 25 July, 2009
I wonder whether I can do similar studies on the drosophila fly larvae by apropriate modifications! I will be grateful if some one tells me the status on fly larval adaptation studies. MC Arunan
Arunan MC
commented on 25 July, 2009
I wonder whether I can do similar studies on the drosophila fly larvae by apropriate modifications! I will be grateful if some one tells me the status on fly larval adaptation studies. MC Arunan
Associated Publications
A trophic role for Wnt-Ror kinase signaling during developmental pruning in Caenorhabditis elegans
by Yu Hayashi, Takaaki Hirotsu, Ryo Iwata, +4
Nature Neuroscience (10 June, 2009)