Figure 5.

Rescue of the learning, but not the 90 min memory deficit of drk^ΔP24 heterozygotes by conditional expression of Ras and Raf transgenes in the MBs. A, MAPK phosphorylation is impaired upon abrogation of DRK in the adult nervous system. A representative semiquantitative Western blot is shown below the quantitative data from four independent blots. The ratio of pMAPK/MAPK in the control Elav/+ strain was arbitrarily set to 1. The mean ± SE are shown. ANOVA indicated significant differences between genotypes (F_(3,19) = 3.853, p < 0.05). Compared with control, phosphorylated MAPK was significantly reduced in Elav/+; drkR-1.2/+; drkR-2/+ (p < 0.01) and drk^ΔP24/+; drkR-2/+ (p = 0.01) while the difference from Elav/+; drk^ΔP24/+; drkR-2/+ was significant at p = 0.02 (Student's t test). B, Performance after 6 CS/US training in flies carrying a Ras^V12S35 effector loop mutant transgene in a mutant background (gray bar) kept inactive before training (uninduced), did not perform better than mutant animals without it (open bars). Mean performance indices and SEMs (PI ± SEM) are shown as in all experiments below. ANOVA indicated genotype effects (F_(4,30) = 5.991, p < 0.0001, n = 6). However, subsequent contrast analysis demonstrated that the differences were among control groups (black bars) and flies carrying the drk^ΔP24 mutation (p < 0.001), and the differences between the later two groups were not significant (p < 0.57). C, Expression of the Ras^V12S35 transgene in the MBs (induced) by inactivation of the Gal80^ts rescued the learning deficit of drk^ΔP24 animals (open bar) as indicated by ANOVA (F_(4,42) = 6.407, p < 0.0001, n ≥ 8). Contrast analysis demonstrated highly significant differences (p < 0.0003) in the performance of animals carrying the drk^ΔP24 mutation, but expressing the transgene (gray bar) and those that do not (open bar), while the performance of the former was not different from that of controls (p < 0.84). D, Induction of constitutively active RAF (Raf^gof) in the adult MBs rescues learning after 6 CS/US training. Performance only after induction of the transgene is shown, as uninduced transgene did not have any effect on the mutant phenotype as shown above for the Ras^V12S35 transgene. ANOVA revealed significant effect of genotype (F_(3,57) = 8.441, p < 0.0001, n ≥ 10) and treatment (F_(1,87) = 34.02, p < 0.0001). Contrast analysis indicated significant differences between drk^ΔP24/+; Raf^gof/+ animals and flies expressing the transgene (p < 0.0002), indicating full rescue of the defect. No differences were observed between flies expressing the transgene and control groups (p < 0.14). E, Partial rescue of drk^ΔP24heterozygote learning deficits upon conditional accumulation of wild-type RAF protein in the MBs. Performances only after transgene induction is shown. ANOVA revealed differences among RAF accumulating strains (F_(3,35) = 20.816, p < 0.0001, n ≥ 7). Subsequent contrast analysis showed significant differences (p < 0.0001) between drk mutant heterozygotes not expressing the Raf^wt transgene and those expressing it under c772. However, the performance of Raf^wt/+; drk^ΔP24/+ (gray bar) was different (p < 0.05) from that of Raf^wt/+and Raf^wt/+; c772/+ animals. F, The drk^ΔP24/+ 90 min memory impairment is not reversed upon conditional expression of constitutively active RAF in the MBs. Only the performance of animals after induction of the transgenes and 6 CS/US training is shown. ANOVA suggested significant differences (F_(3,37) = 10.449, p < 0.0001, n ≥ 8). Subsequent Dunnett's tests using the performance of Raf^gof/+ as control indicated significant differences with drk^ΔP24/+; Raf^gof/+ (p < 0.005) and c772/drk^ΔP24; Raf^gof /TubGAL80^ts (p < 0.0001) suggesting a lack of rescue. (The difference with c772/+; Raf^gof/TubGAL80^ts was not significant, p > 0.7). G, The drk^ΔP24/+ 90 min memory impairment is not reversed upon expression of wild-type RAF in the MBs after 6 CS/US training. ANOVA indicated significant differences (F_(3,27) = 9.926, p < 0.0002, n ≥ 7), and Dunnett's tests indicated that compared with the performance of Raf^wt/+, that of Raf^wt/+; drk^ΔP24/+ was significantly different (p < 0.007) and importantly so was that of Raf^wt/+; c772/drk^ΔP24 (p < 0.0002) indicating lack of rescue. Performances after transgene induction are shown.