Figure 5.

High levels of hyper-PO4 CREB and its ultradian oscillation are associated with LTM formation. A, Paired conditioning-trained (pct) WT w¹¹¹⁸ flies show robust oscillation of hyper-PO4 CREB compared with unpaired conditioning-trained (uct) w¹¹¹⁸ flies. ZT 2 flies were used for olfactory associative training and sampled at different time points after training. The relatively higher level of hyper-PO4 CREB in uct flies at 2 h and 3 h time points results from background oscillation (Fig. 4). A higher level of the 28 kDa CREB isoform is detectable in the nucleus at the 2 h time point (bottom blots are the top blots exposed to the film for a longer time). B, Notch GOF flies N^nd1 and N^nd3 that express high levels of hyper-PO4 CREB show significantly enhanced 24 h LTM. Circadian entrained WT w¹¹¹⁸, N^nd1, and N^nd3 flies (at ZT 2) were incubated at 30°C for 30 min, then at room temperature for 10 min, and subjected to 3× massed olfactory associative training. The graph shows memory that was assessed 24 h later. Data are mean ± SEM; N = 9 for each genotype. ***p < 0.001 (one-way ANOVA, Dunnett's post hoc). C, Flies with the heat shock inducible Notch transgene (hsN) that are known to form better memory (Ge et al., 2004) also exhibit higher levels and ultradian oscillation of hyper-PO4 CREB after a 30 min incubation at 37°C. *Background band that serves as an additional loading control. The same fly equivalents were loaded in each lane in B and C.