Fig. 1.
Spaced training activates crebB transcription. (A) CREBB expression visualized in dissected brains with crebB-Gal4-driven UAS-mCD8::GFP (green), counterstained with DLG-antibody immunostaining (magenta), viewed under a confocal microscope. crebB-Gal4 labels all MB lobes (Center). Optical slices of vertical and horizontal MB lobes (Right) show more prominent expression in α/β neurons than in α’/β’ and γ neurons. (Scale bar, 10 μm.) (B and C) Promotor activation of crebB 24 h after training reported by de novo KAEDE synthesis, estimated by the ratio of new (green, 488 nm) and preexisting (red, 561 nm) proteins (% ∆ F/F¯0). For each brain, single optical slices through the MB α-lobe tip or EB were imaged under identical conditions. (B) Spaced training stimulates crebB activity preferentially in the α-lobe, in comparison with EB controls. (C) A minimum of 5×S training cycles are necessary to observe KAEDE synthesis reflecting crebB activity. Bars represent mean ± SE, n ≥ 8. (D) adult-stage-specific RNAi downregulation of CREBB proteins (–CREBB) in MB α/β neurons impairs 1-d memory after 10×S training (Left). Gal4-targeted RNAi downregulation is induced at the restrictive temperature for tub-Gal80ts (30 °C) from 7 d before training until testing. Memory is unaffected in these flies held at the permissive temperature for tub-Gal80ts (18 °C) after 10×S (Center) and at 30 °C after 10×M (Right). In the same figure, all experiments were done within the same experimental group. Memory performance indices are calculated as the normalized percent avoidance of shock-paired odor. Bars represent mean ± SE, n = 8/bar unless stated otherwise. *P < 0.05; **P < 0.01, ***P < 0.001. All genotypes are listed in SI Appendix, Table S2.