Mean Performance Indices and their Standard Errors of the Mean (PI ± SEM) are shown for all experiments.
(A) Performance immediately after conditioned odor avoidance after training with the indicated number of pairings. n=10 for all points. The performance of ry ⁵⁰⁶flies was significantly different from that of and drk^ΔP24/+ after 6 and 8 (p<0.001- Student's t-test), but not after 12 pairings (p<0.02).
(B) Learning after training with 6 CS/US pairings of drk mutant alleles and controls. n≥12. ANOVA indicated significant effects of genotype (F_{(5, 83)}=13.956, p<0.001) and subsequently confirmed for each allele against the control with Dunnett's tests.
(C) Memory of 6 CS/US training for two representative drk alleles. n≥ 12 for each time point. Time 0 represents learning. Two way ANOVA revealed significant effects of genotype (F_{(2, 218)}= 11.627, p<0.001) and time (F_{(5, 218)} = 18.732, p<0.001).
Subsequent planned comparisons revealed significant differences (p<0.001) between the drk^ΔP24 and drk^EoA heterozygotes and controls at all time intervals, except at 360 minutes where the differences were significant at the p<0.05 level (actual values p=0.016 and p=0.028 for drk^ΔP24 and drk^EoA respectively).
(D) RT-PCR from heterozygous drk deletion mutants carrying either the drkT5.3 or the drkT1 transgenes subjected to three heat shock inductions, or left untreated (0) prior to RNA isolation. Amplification of rp49 transcripts served as a semi-quantitative control. The empty lane displays PCR amplification products from non reverse transcribed RNA.
(E) Learning of drk mutant heterozygotes bearing the drkT5.3, drkT1 transgenes and controls after induction (black bars), or without induction (open bars). n ≥ 10. ANOVA indicated significant effects of treatment (F_{(1, 104)} =15.376, p<0.001) and genotype (F_{(9, 104)}=21.327, p<0.001). Subsequent Tukey-Kramer test (α=0.001) indicated significant differences between induced and uninduced drk^ΔP24/+; T5.3, drk^ΔP24/+; T1and drk^EoA/+; T5.3, but not between heat shocked and not shocked ry⁵⁰⁶, drk^ΔP24/+. In addition, the performance of induced drk^ΔP24/+; T5.3, drk^ΔP24/+; T1and drk^EoA/+; T5.3 was different from that of controls under the same conditions.
(F) Conditional rescue of the 90 minute memory deficit of drk deletion heterozygotes. All strains were treated prior to conditioning as described above, trained, stored at the training temperature (24-25°C) and tested 90 minutes post-training. Group mean PIs ± SEM are shown for n ≥ 10. ANOVA indicated significant effects of treatment (F_{(1, 64)} =21.368, p<0.001) and genotype (F_{(5, 64)} =14.521, p<0.001). Subsequent Dunnett's tests confirmed significant differences in performance of heat shocked ry and drk^ΔP24/+ and non heat shocked drk^ΔP24/+; T5.3 (p<0.001). However the performance of heat shocked drk^ΔP24/+; T5.3 was statistically indistinguishable from that of similarly treated ry and untreated ry flies.

Mean Performance Indices and Standard Errors of the Mean (PI ± SEM) are shown for the indicated genotypes.
(A) Abrogating DRK in the MBs impairs 90-minute memory. Initial training with 8 CS/US produced equal immediate performance between control animals and animals expressing drkRNA-i (F_(2,15)=1.761, p>0.2), n=5. ANOVA for 90-minute memory of the training revealed significant effects of genotype (F_(2,50)=13.490 p<0.0001). n≥15.
(B) Training RNA-i transgene expressing animals with increased number of pairings does not improve the memory impairment, revealing a specific effect of DRK abrogation in the MBs on memory. Learning was equal between under-trained controls (6 pairings) and drkR-2 expressing animals trained with 8 CS/US. (F_(2,12)=0.029, p>0.86, n=6). In contrast, 90-minute memory remained impaired in RNAi-expressing flies compared to under-trained controls (F_(1,46)=18.057, p<0.0001, n≥20).
(C) The 90-minute memory impairment remained unaltered by more intensive training of drk^ΔP24/+ heterozygotes indicating a memory specific impairment. ANOVA did not reveal differences between under-trained controls and drk^ΔP24/+ heterozygotes trained with 8 pairings. (F_(1,12)=2.327, p>0.16, n=6). 90-minute memory was significantly impaired in drk^ΔP24/+ flies compared to under-trained controls (F_(1,16)=5.978, p<0.05, n≥ 7).

(A) Representative western blots probing the level of phosphorylated MAPK (pMAPK) in comparison to total levels of the protein (MAPK) following associative conditioning as detailed in Materials and Methods. The level of Tubulin in each of the indicated control and mutant head lysates was used to normalize protein levels in each lane and for quantification. Lysate from a single head equivalent was loaded per lane. Naïve denotes lysates from w¹¹¹⁸ animals that underwent all manipulations in parallel except for associative learning. Blots from animals allowed to rest 2 minutes, 15 minutes or 90 minutes post-training (as indicated in the graph below n B), followed by lysate preparation are shown left to right.
(B) The mean and its standard error from at least 3 independent determinations of the relative amount of pMAPK to that of MAPK in the samples is shown. The levels of pMAPK and MAPK were determined relative to Tubulin to normalize loading differences. This ratio for naïve animals was fixed to 1. ANOVA did not indicate significant differences among the samples obtained 2 minutes after training (F_(3,19)=1.350, p<0.29, n=4). In contrast, differences among the samples obtained 15 minutes post-training were significant (F_(3,15)=3.998, p<0.03, n=3). Subsequent pairwise t-tests showed significant difference from naive only for wild type flies (p<0.009), but not for drk^ΔP24/+ or drk^E0A/+ (p<0.45 and p<0.29 respectively). Contrast analysis showed that w¹¹¹⁸ pMAPK levels were significantly different from drk^ΔP24/+ and drk^E0A/+ (p<0.02). 90 minutes post-training, ANOVA indicated significant effects of genotype (F_(3,17)= 7.638 p<0.003, n=4). Pairwise t-tests showed that wild type pMAPK levels were not different from that in naïve animals (p<0.16) indicating that MAPK phosphorylation returned to naive levels. In contrast, pMAPK levels in drk^ΔP24/+ and drk^E0A/+ were different (lower) that in naïve flies (p<0.04 and p<0.01 respectively). Contrast analysis also showed that pMAPK levels in w¹¹¹⁸ were significantly higher than those in drk^ΔP24/+ and drk^E0A/+ (p<0.004).
(C) Western blots probing the level of pMAPK in single dissected brains of naïve flies carrying Raf^gof (left side), or Raf^WT (right side) transgenes in control (w¹¹¹⁸), or drk^ΔP24/+ mutant background, with of without transgene induction by incubation at 30°C as indicated by the crosses. The pMAPK level is elevated upon Raf^gof and to a lesser degree Raf^WT expression in w¹¹¹⁸, but not when the flies are heterozygous for the drk^ΔP24 null allele. Quantification of the levels revealed highly significant differences between controls and drk^ΔP24/+ upon Raf^gof (F_{(1, 6)}= 354, p<0.001, n=3) and upon Raf^WT (F_(1,6)= 7.85, p<0.04, n=3) expression.

Immunohistochemical detection of DRK in 5μm frontal (A-D) and sagittal (E-F) paraffin sections through the adult brain. Dorsal is up in all photographs. (A) DRK is absent from MB cell bodies (large arrowhead), while low levels appear in the calyces (ca) barely above general neuropil staining. Arrow points to four positively staining axonal fascicles. (B) DRK accumulates within the pedunculus (p), and R4 neurons of the ellipsoid body (eb arrowhead). (C) Preferential DRK distribution within the α, β and γ (D) lobes. Low level of the protein is detectable in glomeruli of the antennal lobe (al, arrowhead in D). (E and F) Saggital sections demonstrate the differential distribution of DRK within the pedunculus, α, β and γ lobes as indicated by the arrows. Anterior is to the left.

(A) Accumulation of DRK in head lysates from controls, drk^ΔP24/+ and animals driving pan-neuronal expression of drkR-1.2 and drkR-2 simultaneously with Elav. A representative semi-quantitative western blot of such lysates from the indicated genotypes challenged with a-DRK and a-SYNTAXIN as loading control is shown below, while quantification of five independent such experiments is shown above. Means ± SEM are shown. The level of DRK normalized over the level of SYNTAXIN in control strains was arbitrarily set to 1 (Philip et al., 2001). DRK in extracts from heterozygous mutants and elav driven transgenes was statistically significant from that in controls (p<0.001, planned comparisons). No difference was observed between drkR-1.2/+; drkR-2/+ animals controlling for potential effects of the insertions on DRK levels or elav/+ controls (p>0.3).
(B) Reduction of DRK levels in the MBs upon RNA-i transgene expression illustrated in 6 μm paraffin sections challenged with a-DRK antibody. 1,2: drkR-2/+ control animals. 3,4: drk^ΔP24/+; drkR-2/+ mutant heterozygotes. 5,6: c772/+; drkR-2/+. 7,8: drk^ΔP24/c772; drkR-2/+. Sections from all genotypes were processed in parallel and each slide contained control animals and all experimental genotypes. Representative images at the levels of the γ-lobe (1, 3, 5, 7) and α/β lobes (2, 4, 6, 8) are shown. Arrows point to the α lobes on the respective genotypes where the reduction in DRK is most obvious.
(C) Abrogation of DRK in α/β and γ MB lobes with c772 impairs learning and phenocopies the drk mutant phenotype. Group mean PI ± SEMs are shown, n≥10. ANOVA showed significant effects of genotype (F_(3,122)=16.006, p<0.0001) and number of CS/US pairings (F_(2,122)=19.185, p<0.0001). The performance after 8 CS/US was not significantly different in experimental animals with their respective un-driven heterozygous transgene controls (p>0.53). However, with reduced pairings the effects were significant (planned comparisons, p<0.0001)
(D) Abrogation of DRK in α/β MB lobes with c739 impairs learning and phenocopies the drk mutant phenotype. Group mean Performance Indices and their Standard Errors of the Mean (PI ± SEM) are shown, n≥7. ANOVA showed significant effects of genotype (F_(3,88)=13.282, p<0.0001) and number of pairings (F_(2,88)=19.168, p<0.0001). The performance after 8 CS/US was not significantly different in experimental animals with their respective undriven heterozygous transgene controls (p>0.56). However, with reduced pairings the effects were significant (planned comparisons, p<0.0001)
(E) The learning impairment is dependent on the amount of DRK protein within the MBs. PI ± SEMs are shown, n≥9. ANOVA indicated significant effects of genotype F_(4,56)=14.878, p<0.0001. Subsequent contrast analysis showed significant differences in the performances of drkR-1.2/+; drkR-2/+ control animals (black bars) and c772/+; drkR-2/+ (p<0.0005). The performance of c772/+; drkR-2/+ was also significantly different from that of drk^ΔP24/c772; drkR-2/+ (p<0.001), and from drkR-1.2/c772; drkR-2/+ (p<0.05).
(F) Abrogation of DRK only within the MBs impairs learning. PI ± SEMs are shown, n≥9. ANOVA revealed significant effects of genotype F_(4,57)=17.480, p<0.0001. Planned comparisons did not reveal any differences between drkR-1.2/+; drkR-2/+ controls 2/+, 772/+ and drkR-2/c232 (p>0.06). The differences between controls and c772/+; drkR-2/+ and drk^ΔP24/+; drkR-2/+ remained significant p<0.0001).

(A) MAPK phosphorylation is impaired upon abrogation of DRK in the adult nervous system. A representative semi-quantitative 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 ± its standard error are shown. ANOVA indicated significant differences between genotypes (F_(3,19)=3.853, p<0.05). Compared to 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 prior to training (uninduced), did not perform better than mutant animals without it (open bars). Mean Performance Indices and Standard Errors of the Mean (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 than 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-minute 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-minute 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 to 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.