Time requirements for synaptic output from MBN subsets for the retrieval of appetitive olfactory memory. A, All flies were conditioned using an appetitive unconditioned stimulus at 24°C and shifted to 32°C 10 min before a retrieval test. A synaptic blockade imposed by the expression and activation of shits in the α′/β′ MBNs significantly reduced memory expression from 15 min to 3 h after training (Mann–Whitney pairwise comparisons, p ≤ 0.0022). No significant difference was observed during retrieval of memory from 9 to 24 h 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, Synaptic blockade of γ MBNs significantly reduced appetitive olfactory memory expression from 15 min to 3 h after training (Mann–Whitney pairwise comparisons, p ≤ 0.0411). No significant difference was observed during retrieval of memory from 9 to 24 h 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, Synaptic blockade of α/β MBNs significantly reduced appetitive olfactory memory expression from 15 min to 24 h 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, Synaptic blockade using shits of α/β and γ MBNs eliminated appetitive olfactory memory expression from 15 min to 24 h 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 significantly 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).