Figure 1.

Aversive Olfactory Learning with DEET Reinforcement

(A) Learning with DEET depends on concentration. Wild-type flies starved overnight were trained with 0%–0.8% DEET with a sugar carrier (3 M xylose plus 100 mM sucrose). Aversive memory performance increased with DEET concentrations up to 0.4% but was negligible with 0.8% DEET. Learning with 0.4% DEET is significantly different from that with both 0.1% and 0.8% DEET (both p < 0.001). All other group wise comparisons are not significant (all p > 0.05). ANOVA followed by Tukey’s multiple comparison test was performed. p values are multiplicity adjusted (ANOVA). n ≥ 5.

(B) DEET inhibits ingestion. The amount of sugar carrier plus DEET (0.4% or 0.8%) ingested in 5 min was quantified using FD&C Blue No. 1 dye supplementation. Flies consumed sugar carrier alone, but inclusion of 0.4% or 0.8% DEET significantly reduced consumption. However, flies consumed statistically significant amounts of dye presented with 0.4% DEET, but not with 0.8% DEET (p = 0.002 and p = 0.8427, respectively, from zero; one-sample t test, n ≥ 5).

(C) DEET learning is most robust when flies ingest. Hungry flies display robust immediate aversive memory with 0.4% DEET presented with sugar carrier. However, both satiated flies and those trained with 0.4% DEET without sugar carrier exhibited significantly less aversive memory performance (both p < 0.05, ANOVA, n ≥ 10).

(D) DEET memory is labile. DEET reinforced memory decayed rapidly and was not significant 15 min after training (p < 0.05 versus 3 min performance and p > 0.9 versus 30 and 60 min, ANOVA, n ≥ 6).

(E) Ablation of bitter-taste neurons impairs DEET learning. Flies expressing UAS-hid and UAS-rpr in Gr66a-GAL4 cells were trained with 0.4% DEET in sugar carrier (3 M xylose and 100 mM sucrose) and were immediately tested for memory performance. The performance of these flies was statistically different than that of the control groups (p < 0.05, ANOVA, n ≥ 23).

(F) Aversive memory can be implanted with bitter-taste neuron activation contingent with odor presentation. Flies were trained by pairing dTrpA1-mediated activation of Gr66a-GAL4 bitter gustatory neurons with odor as shown in the schematic. Significant aversive memory was formed in Gr66a-GAL4/UAS-dTrpA1 flies that persisted for at least 3 hr (at both time points p < 0.005, ANOVA, n ≥ 8).

Data are shown as mean ± SEM. Asterisks denote significant difference between marked group and the relevant controls. See also Figure S1.