Fig. 3

Ank2 is required for long-term memory. Learning and memory were assessed with the courtship suppression assay. The controls included in each assay are the GAL4 driver (plus tubP-GAL80ts where indicated) crossed to CS, and UAS-Ank2 RNAi1 crossed to CS, such that the progeny are heterozygous for either the driver or the RNAi. (A-D) elav-GAL4 and UAS-Ank2 RNAi1 flies were crossed to achieve pan-neuronal knockdown of Ank2 in progeny. (A) Learning was unaffected by knockdown of Ank2 (ANOVA, F_(2,47) = 0.002, p = 0.252). (B) Immediate memory was also unaffected (ANOVA, F_(2,45) = 0.044, p = 0.819). (C) Ank2 knockdown impaired long-term memory (ANOVA, F_(2,60) = 7.31, p < 0.001; post-hoc Tukey’s HSD, **p < 0.01). (D) Courtship activity was not impaired by pan-neuronal knockdown of Ank2 (ANOVA, F_(2,51) = 0.14, p = 0.870). (E-N) Knockdown of Ank2 in the adult mushroom body impairs LTM. Ank2 was knocked down in specific regions of the brain by crossing Ank2 RNAi to the indicated driver line and tubP-Gal80ts. (E) Schematic diagram depicting the induction of Ank2 RNAi expression in the adult mushroom body. Expression was restricted to the adult brain by raising flies at raised at 19°C, at which temperature GAL80 represses GAL4. After eclosion, when flies were 3–5 days old, the temperature was raised to 30°C for 72 hours, after which training commenced. At this temperature GAL80 is inactivated, allowing GAL4 to induce transgene expression. Twenty-four hours after training, the flies were equilibrated to 25°C for one hour prior to testing. (F) Schematic diagram labelling the lobes of the mushroom body in which Ank2 was knocked down. (G) Pan-neuronal knockdown of Ank2 in the adult brain impaired long term memory (ANOVA, F_(2,54) = 0.317, p < 0.01; post-hoc Tukey’s HSD, *p < 0.05). (H) Similarly, memory was also impaired when knockdown of Ank2 was restricted to the mushroom body (ANOVA, F_(2,52) = 0.922, p < 0.001; post-hoc Tukey’s HSD, **p < 0.01, *p < 0.05). (I) When knockdown was restricted to the α/β and γ neurons of the mushroom body, long-term memory was still disrupted (ANOVA, F_(2,51) = 0.923, p < 0.0001; post-hoc Tukey’s HSD, **p < 0.01). (J) Reduction of Ank2 in just the α/β neurons had no significant effect on long-term memory (ANOVA, F_(2,41) = 0.025, p = 0.819). (K) There was also no impairment when Ank2 was reduced in the α’/β’ neurons (ANOVA, F_(2,51) = 0.122 p = 0.372). (L) Ank2 is required in the γ lobes as knockdown with NP1131-GAL4 impaired LTM (ANOVA, F_(2,46) = 0.312, p < 0.01; post-hoc Tukey’s HSD, *p < 0.05). (M) A second γ lobe driver R16A06-GAL4 crossed to Ank2 RNAi2 also impaired LTM significantly (ANOVA, F_(2,33) = 18.57, p < 0.0001, post-hoc Tukey’s HSD, **p < 0.01). (N) A weaker γ lobe driver 1471-GAL4 also reduced LTM, however this was not quite significant (ANOVA, F_(2,59) = 0.210, p = 0.056)