Figure 5. Drep-2 is required in KCs for olfactory short- and intermediate-term memory.

(A) Flies mutant for drep-2 sense electric shock and the odors 4-methyl-cyclohexanol (4-MCH) and 3-octanol (3-OCT) normally; there is no difference in mean performance indices between mutants and isogenic w¹¹¹⁸ control flies (Mann–Whitney U tests (MWU)). Sample sizes n are indicated with white numbers; grey bars show SEMs. (B) Both mutants drep-2^ex13 and drep-2^ex27/Df^w45-30n are deficient in aversive olfactory conditioning, 3 min STM in a T-maze. The graph shows mean learning indices and SEMs. Mutants performed significantly worse than isogenic controls (MWU: p = 0.00001 for both comparisons, Bonferroni-corrected significance level α = 0.0167, 3 tests). (C) Re-expression of drep-2 cDNA with elav^III-Gal4 (pan-neural), 30y-Gal4 (MB KCs), or mb247-Gal4 (MB KCs) restores the deficit to normal levels. Heterozygous drep-2^ex13 mutants do not display a significant deficit. MWU for individual comparisons showed a significant difference between these groups (α = 0.0042, 12 tests): w¹¹¹⁸ and drep-2^ex13 (p < 0.00001), drep-2^ex13/drep-2^ex13 and drep-2^ex13/+ (p < 0.00001), drep-2^ex13 and drep-2^ex13;uas-drep-2/elav^III-gal4 (p < 0.00001), drep-2^ex13 and drep-2^ex13;uas-drep-2/30y-gal4 (p < 0.00001), drep-2^ex13 and drep-2^ex13;uas-drep-2/mb247-gal4 (p < 0.00001). None of the differences indicated as not significant had a p < 0.12, except for w¹¹¹⁸ and drep-2^ex13/+ (p = 0.03851; not significant in the case of α = 0.0042). (D) Intermediate-term memory (ITM = ASM + ARM) performance. Mutants (drep-2^ex13) are defective in ASM, but not in ARM. The defect can be restored with elav^III-Gal4 or mb247-Gal4 (30y-Gal4 was not used here). Statistical tests were run separately for ITM and ARM. For ITM, MWU for individual comparisons showed a significant difference between these groups (α = 0.00625, 8 tests): w¹¹¹⁸ and drep-2^ex13 (p < 0.0001), drep-2^ex13 and drep-2^ex13;uas-drep-2/elav^III-gal4 (p < 0.0001), drep-2^ex13 and drep-2^ex13;uas-drep-2/mb247-gal4 (p < 0.0001). For assessing differences in ARM, ITM and ARM performances of each genotype were compared with MWU. The following genotypes showed a significant difference between ITM and ARM (α = 0.0071, 7 tests): w¹¹¹⁸ (p < 0.0001), drep-2^ex13;uas-drep-2/elav^III-gal4 (p = 0.0002), drep-2^ex13;uas-drep-2/mb247-gal4 (p = 0.0006). None of the differences indicated as not significant had a p < 0.11.

DOI: http://dx.doi.org/10.7554/eLife.03895.010

Figure 5—figure supplement 1. PN-KC synapses appear morphologically normal in drep-2 mutants.

(A) Absence of major neuroanatomical defects in drep-2^ex13 mutant brains. MB lobes, Fasciclin II (FasII) staining, maximum intensity projections. Scale bar: 10 μm. (B) Antibody staining of w¹¹¹⁸ control and drep-2^ex13 mutant brains, using antibodies against the postsynaptic ACh receptor subunit Dα7 and presynaptic Brp^N−Term. Focus on microglomeruli of PN-KC synapses in the MB calyx. Microglomeruli of mutants appear structurally normal. Scale bar: 1 µm. (C) Electron microscopy of w¹¹¹⁸ control and drep-2^ex13 mutant brains. Microglomeruli and postsynaptic KC profiles of mutants appear structurally normal. Scale bars: 100 nm. (D) The number of synapses (active zones) in the MB calyx does not significantly differ between drep-2^ex13 mutants and w¹¹¹⁸ controls. Syd-1-positive spots were counted and compared between genotypes as described (Kremer et al., 2010). No significant difference was found between the number of spots (MWU, p = 0.62). Average synapse counts were in the range expected (28,000–30,000 [Kremer et al., 2010]).

DOI: http://dx.doi.org/10.7554/eLife.03895.011