We further challenged a valence-specific role for αβ neuron subsets

using additional genetic approaches. We first confirmed that αβs neurons are required for both appetitive and aversive memory retrieval using NP5286, another LGK-974 in vivo GAL4 line with strong expression in αβs neurons and weaker expression in αβp neurons (Figures 3A and S1F; Tanaka et al., 2008). Appetitive and aversive memory performance of NP5286;shits1 flies was statistically different to that of shits1 and GAL4 control flies ( Figures 3E and 3F). No statistical differences were apparent when experiments were performed at permissive 23°C ( Figure S3) and the NP5286;shits1 flies exhibit normal olfactory acuity at the restrictive temperature ( Table S1). We next challenged an appetitive memory-specific role for αβc neurons using an intersectional genetic strategy. Combining a ChaGAL80 transgene with c739 removes expression in the αβs and αβp neurons from the c739-labeled αβ population and leaves robust expression in αβc neurons (Figures 3B and S1G). We again trained flies at the permissive Metabolisms tumor temperature and blocked αβc during retrieval. Similar to the analysis with NP7175;shits1

flies, appetitive memory performance of c739;ChaGAL80/shits1 flies was impaired, being statistically different to the relevant control groups ( Figure 3E). Moreover, the c739 disruptive effect on aversive memory was abolished with ChaGAL80, consistent with removal of αβs expression from c739 ( Figure 3F). Control experiments at 23°C did not reveal significant differences between the relevant groups ( Figure S3). A role for αβc in memory consolidation has been reported (Huang et al., 2012). Blocking NP6024-labeled αβc neurons for several hours after training disrupted appetitive and aversive memory consolidation, whereas blocking NP7175-labeled neurons only impaired aversive memory consolidation

(Huang et al., 2012). Although others defined the αβ neurons labeled in NP6024 as inner and outer αβc neurons (Tanaka et al., 2008 and Huang et al., 2012), our anatomical analysis revealed that outer αβc neurons occupy the area of the vertical MB lobe that is anatomically indistinguishable from that containing oxyclozanide the αβs neurons (Figures 3A–3D). Furthermore, blocking output during retrieval in NP6024;shits1 flies significantly impaired both appetitive and aversive memory ( Figures 3E and 3F), consistent with NP6024 expressing in αβc and αβs neurons. Control experiments at the permissive temperature did not reveal significant differences between the relevant groups ( Figure S3) and the NP6024;shits1 flies exhibit normal olfactory acuity ( Table S1). Perhaps more importantly, a consolidation effect cannot account for our appetitive memory retrieval-specific function because the retrieval role for αβc is not time dependent.