The Long-Term Memory Trace Formed in the Drosophila α/β Mushroom Body Neurons Is Abolished in Long-Term Memory Mutants

Long-term memory in 26 LTM mutants. A, An initial screen of mutant flies carrying one copy of c739-Gal4 and one copy of Uas-G-CaMP was performed using a 5× spaced conditioning protocol. Flies received forward conditioning with 1 min exposure to the CS+ odor (Ben) with 12 electric shock pulses (90 V), followed by 1 min exposure to the CS− odor (Oct) without electric shock after a 30 s exposure to fresh air. The training protocol was performed a total of five times with a 15 min intertrial interval (ITI). Flies were transferred to a T-maze at 24 h after conditioning and tested for behavioral memory. Naive groups of flies were carried through the same manipulations as the conditioned animals except that they were not exposed to odor and electric shock. Each conditioned group of flies was tested in parallel with a naive group. Some flies were separated before behavioral testing and analyzed for cellular memory by functional imaging (Fig. 2). The performance gains of the trained flies were measured as ΔPI. This was computed by subtracting the score of a naive group of flies from the corresponding conditioned group. In all cases, we chose experiments wherein the average scores of naive animals for each group were not statistically significant (Wilcoxon test) from zero to prevent floor and ceiling effects. For the c739; Uas-G-CaMP control flies, spaced conditioning was performed a total of three times across the several months that this experiment was conducted with an n = 6 for each experiment. The 24 h memory scores for control groups (∼0.4) were within the normal range of LTM scores we have come to expect and comparable to the LTM scores obtained by differential conditioning procedures that average the performance gains using two different CS+ odors (Tully et al., 1994; Perazzona et al., 2004; Yu et al., 2006). The ΔPI values were subjected to nonparametric tests; i.e., a Kruskal–Wallis test for multiple comparisons with genotype as the main factor, Mann–Whitney U test for comparing two independent samples, and Wilcoxon matched pairs test to compare single performance indices against zero. p values were corrected for the multiple comparisons to the control using the Benjamini and Hochberg false discovery rate. Out of the 26 lines tested, 22 yielded p values that were significantly different from the control (Kruskal–Wallis statistic 64.67, p < 0.0001; corrected Mann–Whitney pairwise comparisons, p ≤ 0.023). One mutant (E3803) was marginally nonsignificant (corrected Mann–Whitney pairwise comparison, p ≤ 0.051). This mutant was not selected for retesting given the marginally nonsignificant probability and because this is a replication experiment of Dubnau et al. (2003). The mutants C0150, E0627, and c0167 yielded p values that were not significantly different from the control (corrected Mann–Whitney pairwise comparisons, p ≥ 0.136. n = 12 for all groups). Error bars are the SEM. B, A second test of the three LTM mutants that failed to reach significance. Flies from each mutant genotype (C0150, E0627, and c0167) were trained using the 5× spaced conditioning protocol and compared to the c739; Uas-G-CaMP control. In each case, the mutant group performed significantly differently from its corresponding control group (Mann–Whitney pairwise comparisons, p ≤ 0.0433; n = 12 for all groups). Significance levels are indicated (*p ≤ 0.05, **p ≤ 0.01). Error bars are the SEM.