A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Summary

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Abstract

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann^1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor³ for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,⁴ uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC⁴. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC⁵. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC^6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

Protocol

1. Construction of GFP Tagged EGL-4 Expressing Animals

1. Clone the translational fusion GFP::EGL-4 under the promoter for the odr-3 gene (use 2678 bp directly upstream of the start codon): (p)odr-3::GFP::EGL-4. The odr-3 promoter drives expression in the amphid neuron pairs: AWA; AWB; AWC; and weakly in ASH.
2. Inject the plasmid (p)odr-3::GFP::EGL-4 into wildtype (N2) animals at 50 ng/μl with the co-injection markers using standard germ-line transformation techniques⁸: (p)ofm-1::GFP⁹ (25 ng/μl), and (p)odr-1::dsRed¹⁰ (25 ng/μl). The odr-1 promoter....

Results

An example of the localization pattern of GFP::EGL-4in the AWC before and after prolonged odor exposure is shown in Figure 2. Prior to prolonged odor exposure, GFP::EGL-4 is localized to the cytosol of the AWC (Figure 2B), and after 80 min odor exposure GFP::EGL-4 is localized to the nucleus of the AWC (Figure 2D). At the behavioral level, animals with cytosolic GFP::EGL-4 in AWC are attracted to a point source of odor (Figure 2C graphs representative re.......

Discussion

The odor-induced nuclear entry of a GFP-tagged EGL-4 molecule described here provides a robust molecular readout of olfactory adaptation in C. elegans. The odor-induced nuclear translocation assays are straightforward and require only a few days of preparation time. The pyIs500 animal that we have constructed for these assays, expresses a marker that illuminates the AWC neuron as well as expressing the GFP-tagged EGL-4 protein. Thus, we feel that an experimenter with little to no experience working with.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Agar	Difco	DF0140-07-4	NGM plates
Sodium Chloride	Fisher Chemical	S671-10	NGM plates
Bacto Peptone	Difco	DF0118-07-2	NGM plates
Potassium Phosphate Dibasic	Fisher Chemical	S375-500	S-Basal buffer and NGM plates
Potassium Phosphate Monobasic	Fisher Chemical	P285-500	S-Basal buffer and NGM plates
Kimwipes - Small	Kimberly-Clark	LS2770
Ethanol 100%	Gold Shield Chemical Co.	43196-115	diluting odors for chemotaxis assays
Calcium Chloride	Sigma-Aldrich	C8106-500G	NGM plates
Magnesium Sulphate	MP Biomedicals	150136-500G	NGM plates
Sodium Azide 99%	Fisher Scientific	ICN10289180	Anesthetic
Agarose - UltraPure	Invitrogen	16500-500	Agarose pads
Benzaldehyde	Sigma-Aldrich	B1334-100G	AWC odor
Butanone, ACS Grade	Sigma-Aldrich	360473-500ML	AWC odor
Microcentrifuge Tubes - 1.5 ml Colored	Denville	LS8147
Pasteur Pipet Disposable Glass 5-3/4"	Fisher Scientific	13-678-20B
Stratalinker	Stratagene	Stratalinker 2400	UV integration
Filter Vacuum Bottle - 500 ml	Nalgene	09-740-25B