Time requirements for synaptic output from MBn subsets for the retrieval of appetitive olfactory memory. (A) All flies were conditioned using an appetitive US at 24°C and shifted to 32°C ten minutes prior to a retrieval test. A synaptic blockade imposed by the expression and activation of shits in the α′/β′ MBn significantly reduced memory expression from 15 min to 3 hr after training (Mann-Whitney pairwise comparisons, p ≤ 0.0022). No significant difference was observed during retrieval of memory from 9 to 24 hr after training (Mann-Whitney pairwise comparisons, p ≥ 0.2403). No significant difference was observed between permissive and restrictive temperatures for control flies carrying only the c305a-gal4 element (Mann-Whitney pairwise comparisons, p ≥ 0.5887). (B) A synaptic blockade of γ MBn significantly reduced appetitive olfactory memory expression from 15 min to 3 hr after training (Mann-Whitney pairwise comparisons, p ≤ 0.0411). No significant difference was observed during retrieval of memory from 9 to 24 hr after training (Mann-Whitney pairwise comparisons, p ≥ 0.3315). No significant difference was observed between permissive and restrictive temperatures for control flies carrying only the 1471-gal4 element (Mann-Whitney pairwise comparisons, p ≥ 0.4696). (C) A synaptic blockade α/β MBn significantly reduced appetitive olfactory memory expression from 15 min to 24 hr after training (Mann-Whitney pairwise comparisons, p ≤ 0.045). No significant difference was observed between permissive and restrictive temperatures for control flies carrying only the c739-gal4 element (Mann-Whitney pairwise comparisons, p ≥ 0.1727). (D) A synaptic blockade using shits of α/β and γ MBn eliminated appetitive olfactory memory expression from 15 min to 24 hr after training (Mann-Whitney pairwise comparisons, p ≤ 0.0304). The performance of 247-gal4/uas-shits flies at restrictive temperature at all times tested was not significant different from zero (Wilcoxon signed rank tests, p ≥ 0.0938). No significant difference was observed between permissive and restrictive temperatures for control flies carrying only the 247-gal4 element (Mann-Whitney pairwise comparisons, p ≥ 0.1087). (E) No significant difference was observed between permissive and restrictive temperatures for control flies carrying only the uas-shits element (Mann-Whitney pairwise comparisons, p ≥ 0.0916).