3 octanol oct

Groups of approximately 20 female flies, 4–10 day post-eclosion, were trained and tested at 25°C at 50% relative humidity in the fully automated olfactory arena for optogenetics experiments (Aso and Rubin, 2016 (link); Pettersson, 1970 (link); Vet et al., 1983 (link)). The odors were diluted in paraffin oil (Sigma–Aldrich): 3-octanol (OCT; 1:1000; Merck) and 4-methylcyclohexanol (MCH; 1:750; Sigma–Aldrich). Videography was performed at 30 frames per second and analyzed using Fiji (Schindelin et al., 2012 (link)). Statistical comparisons were performed (Prism; Graphpad Inc, La Jolla, CA 92037) using the Kruskal Wallis test followed by Dunn's post-test for multiple comparison, except those in Figures 4C, E, 6B, C and E and E, 9A, Figure 4—figure supplement 1 and Figure 5—figure supplement 3C, which used Wilcoxon signed-rank test with Bonferroni correction to compare from zero. Appropriate sample size for olfactory learning experiment was estimated based on the standard deviation of performance index in previous study using the same assay (Aso and Rubin, 2016 (link)). We set the effect size, power and significance as 0.15, 0.8 and 0.05, respectively.

Olfactory learning assays were performed using the four-field optogenetic olfactory arena as previously described (Aso and Rubin, 2016 (link)) using thirty 1 s pulses of red LEDs for activation (627 nm peak and 34.9 µW/mm²). For testing the conditioned response in the presence of DAN activation 30 times of 1 s pulses of red light ON and 1 s OFF were delivered spread over the 60 s test period. Crosses of split-GAL4 lines for DANs, MB043C and MB630B (Aso et al., 2014a (link); Aso and Rubin, 2016 (link)), and 20xUAS-CsChrimson-mVenus in attP18 (Klapoetke et al., 2014 (link)) were kept on standard cornmeal food supplemented with retinal (0.2 mM all-trans-retinal prior to eclosion and then 0.4 mM) at 22°C at 60% relative humidity in the dark. Female flies were sorted on cold plates at least 1 d prior to the experiments and 4–10 d old flies were used for experiments. Groups of approximately 20 females were trained and tested at 25°C at 50% relative humidity in a dark chamber. The odors were diluted in paraffin oil (Sigma–Aldrich): 3-octanol (OCT; 1:1000; Merck) and 4-methylcyclohexanol (MCH; 1:1000; Sigma–Aldrich). For appetitive memory assays using MB043C, flies were starved for 48 hr on 1% agar. Videography was performed at 30 frames/s and analyzed using Fiji (Schindelin et al., 2012 (link)). Statistical comparisons were performed using Prism (Graphpad Inc, La Jolla, CA 92037).