Figure 1.

Schematic of the experimental design and validation of courtship conditioning to induce LTM. A) Long-term memory (LTM) was induced in flies using a previously established seven-hour courtship conditioning protocol (Koemans et al. 2017). Following training, MB-unc84 flies expressing unc84::GFP in the α/β and γ lobes of the MB were collected for INTACT and downstream transcriptome analysis at two time points: one hour and 24 hr after-rejection (AR). RNA was then isolated from the WH and MB-fractions, cDNA libraries prepared, and next-generation sequencing performed. B) Confocal projection of the whole brain showing the expression of UAS-unc84::GFP using R14H06-GAL4. Only a few nuclei outside of the MB calyx are labeled with GFP. MB calyx is indicated. Counterstaining was performed using the nc82 antibody against Bruchpilot. C) Confocal projection showing expression of UAS-mCD8::GFP with R14H06-GAL4 in the α/β and γ lobes of the MB. D) To provide evidence of normal memory in MB-unc84, a subset of flies were collected in parallel with flies used for transcriptome analysis and tested in the courtship conditioning assay. MB-unc84 flies show a decrease in courtship index (CI) at 24h-AR compared to Naïve flies, suggesting normal memory function in this genotype (n = 23 and n =29, respectively, for naïve and trained flies; **** P < 0.001 Mann-Whitney U-test).