Go contributes to olfactory reception in Drosophila melanogaster

Abstract

Background: Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology and function as ligand-gated cation channels. Consequently, the involvement of cyclic nucleotides and G proteins in insect odor reception is controversial. Since the heterotrimeric Goalpha subunit is expressed in Drosophila olfactory receptor neurons, we reasoned that Go acts together with insect odorant receptor cation channels to mediate odor-induced physiological responses.

Results: To test whether Go dependent signaling is involved in mediating olfactory responses in Drosophila, we analyzed electroantennogram and single-sensillum recording from flies that conditionally express pertussis toxin, a specific inhibitor of Go in Drosophila. Pertussis toxin expression in olfactory receptor neurons reversibly reduced the amplitude and hastened the termination of electroantennogram responses induced by ethyl acetate. The frequency of odor-induced spike firing from individual sensory neurons was also reduced by pertussis toxin. These results demonstrate that Go signaling is involved in increasing sensitivity of olfactory physiology in Drosophila. The effect of pertussis toxin was independent of odorant identity and intensity, indicating a generalized involvement of Go in olfactory reception.

Conclusion: These results demonstrate that Go is required for maximal physiological responses to multiple odorants in Drosophila, and suggest that OR channel function and G-protein signaling are required for optimal physiological responses to odors.

Figure 1

PTX reduces the amplitude of ethyl acetate induced EAG responses. EAG traces evoked by the application of 10^-4 ethyl acetate in Gal80^ts20/Or83b-Gal4; UAS-PTX/+ flies at temperatures that restrict (18°C, black lines) or permit (32°C, gray lines) PTX expression.

Figure 2

Inhibition of heterotrimeric G_o signaling reversibly reduces the amplitude of ethyl acetate evoked EAG responses. For all fly strains, a 10^-4 dilution of ethyl acetate was used to evoke EAG responses. (A) EAG responses from Gal80^ts20/+; UAS-PTX/+ and Or83b-Gal4/+ control strains do not decrease (p > 0.5) at 32°C compared to 18°C. Gal80^ts20/Or83b-Gal4; UAS-PTX/+ flies have a significantly (p < 0.0001) higher EAG amplitude in the absence of PTX expression before heat induction (18°C) or after recovery from heat induction (18°C#) than in the presence of PTX expression (32°C). (B) EAG responses from Or83b-Gal4; UAS-rtTA/Tet^o-PTX flies are significantly (p < 0.0001) higher in the absence of PTX expression (dox -) than in the presence of PTX expression (dox +). EAG responses from flies that express PTX-insensitive G_o (PiG_o) in ORNs (Tet^o-PTX, UAS-PiG_o/+; Or83b-Gal4, UAS-rtTA) are not different (p > 0.7) whether PTX expression in ORNs is induced (dox +) or uninduced (dox -). For each genotype and treatment, at least 12 EAG recordings from minimum 6 flies were analyzed. Asterisks denote a significant (p < 0.05) change. All values are mean ± S.E.M.

Figure 3

G_o activity is required for the perdurance of EAG responses. For each fly strain, a 10^-4 dilution of ethyl acetate was used to evoke EAG responses. The EAG fall time constant in Gal80^ts20/+; UAS-PTX/+ and Or83b-Gal4/+ control strains is not different (p > 0.8) at 18°C and 32°C. Gal80^ts20/Or83b-Gal4; UAS-PTX/+ flies have a significantly (p < 0.01) longer EAG fall time constant in the absence of PTX expression (18°C) than in the presence of PTX expression (32°C). For each genotype and temperature, at least 8 EAG recordings from minimum 4 flies were analyzed. Asterisks denote a significant (p < 0.05) change. All values are mean ± S.E.M.

Figure 4

G_o inhibition reduces odor-evoked firing frequency. For each fly strain, CO₂ and a 10^-4 dilution of ethyl acetate were used to evoke spike activity from ab1C or ab1A neurons respectively. Spike frequency in Gal80^ts20/+; UAS-PTX/+ and Or83b-Gal4/+ control strains do not decrease (p > 0.5) at 32°C compared to 18°C. Gal80^ts20/Or83b-Gal4; UAS-PTX/+ flies have a significantly (p < 0.0001) lower ethyl acetate evoked ab1A spike frequency in the absence of PTX expression (18°C) than in the presence of PTX expression (32°C), whereas CO₂-induced single unit responses in the ab1C neuron was not unaffected (p > 0.8). For each genotype and temperature, responses from at least 8 ORNs from minimum 4 flies were analyzed. Asterisks denote a significant (p < 0.05) change. All values are mean ± S.E.M.

Figure 5

G_o signaling is required for normal EAG responses to diverse odorants. (A) EAG responses evoked by four different concentrations of ethyl acetate (EA) in Gal80^ts20/Or83b-Gal4; UAS-PTX/+ flies are significantly (p < 0.0001) higher in the absence of PTX expression (18°C) than in the presence of PTX expression (32°C). (B) EAG responses evoked by a 10^-4 concentration of ethyl acetate (EA), a 10^-4 concentration of isoamyl acetate (IAA), a 10^-4 concentration of cyclohexanone (CH), a 10^-4 concentration of 4-methylcylcohexanol (MCH), and a 10^-3 concentration n-butanol (BUT) in Gal80^ts20/Or83b-Gal4; UAS-PTX/+ flies are significantly (p < 0.0001) higher in the absence of PTX expression (18°C) than in the presence of PTX expression (32°C). For a given genotype, odor concentration, and temperature, at least 8 EAG recordings from minimum 4 flies were analyzed. Asterisks denote a significant (p < 0.05) change. All values are mean ± S.E.M.