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. Author manuscript; available in PMC: 2020 Apr 28.
Published in final edited form as: Nat Neurosci. 2019 Oct 28;22(12):2029–2039. doi: 10.1038/s41593-019-0515-z

Extended Data Fig. 4. Controls and additional experiments related to Figure 4.

Extended Data Fig. 4

Knockdown of lk in all LK neurons (LK-GAL4) or only LHLK neurons (ap-GAL4) using a second RNAi line impairs 6 h sugar memory performance in hungry flies (LK-GAL4: p<0.0001; ap-GAL4: p<0.0025; n = 8; one-way ANOVA with Tukey’s test). b, RNAi knockdown of lk in all LK neurons (LK-GAL4) or only LHLK neurons (ap-GAL4) does not affect immediate sugar memory performance (LK-GAL4: p>0.98; ap-GAL4: p>0.81; n = 8; one-way ANOVA with Tukey’s test). c, Permissive 23°C control for experiments in Fig. 4c. No effect was observed (LK-GAL4: p>0.68; ap-GAL4: p>0.66; n = 8; one-way ANOVA with Tukey’s test). d, RNAi knockdown of Lkr in PAM-β′2a (MB087C-splitGAL4), PPL1-γ2α′1 (MB296B-splitGAL4), or PPL1-γ1pedc (MB320C-splitGAL4) DANs does not affect 6 h sugar memory performance (MB087C: p>0.49, n = 8; MB296B: p>0.98; MB320C: p>0.7; n = 8; one-way ANOVA with Tukey’s test). e, RNAi knockdown of Lkr in DDC-GAL4- but not TH-GAL4-labeled DANs impairs 6 h sugar memory performance in hungry flies (DDC-GAL4: p<0.036, n = 8; TH-GAL4: p>0.97, n = 8; one-way ANOVA with Tukey’s test). f, Adult brain expression of UAS-mCD8::GFP driven by MB056B-splitGAL4 (PAM-β′2mp, green) and LexAop-rCD2::RFP driven by MB247-LexA::p65 (mushroom bodies, magenta). Counterstained with anti-Brp antibody (gray). Four brains were examined and show the same expression pattern. Scale bar 50 μm. g, Knockdown of Lkr in PAM-β′2mp DANs with MB056B-splitGAL4 and a second RNAi line impairs 6 h sugar memory (p<0.0002, n = 8; one-way ANOVA with Tukey’s test). h, Permissive 23°C control for experiments in Fig. 4f. No effect was observed (p>0.079, n = 8; one-way ANOVA with Tukey’s test). i, Blocking PAM-β′2mp (MB056B) DANs with UAS-Shits1 at 32°C 20 min before and during training does not affect 6 h sugar memory performance (p>0.437, n = 8; one-way ANOVA with Tukey’s test). j, Blocking PAM-β′2mp (MB056B) DANs with UAS-Shits1 at 32°C immediately after training until 30 min before testing does not affect 6 h sugar memory performance (p>0.21, n = 8; one-way ANOVA with Tukey’s test). k, Permissive 23°C control for experiments in Fig. 4g. No effect was observed (p>0.55, n = 8; one-way ANOVA with Tukey’s test). l, Adult brain expression of JFRC2-10XUAS-IVS-mCDGFP driven by R67C06-GAL4 (putative Lkr expressing neurons, green). Counterstained with anti-Brp antibody (gray). Confocal stack downloaded from JFRC FlyLight database38. Illustration below indicates position of R67C06 enhancer in fly genome. Scale bar 50 μm. Temperature regimens shown above a-c and g-k. Box-plots: center line indicates median; box limits, upper and lower quartiles; whiskers, max to min range; dots, individual data points. See Supplementary Table 3 for statistics details.