Figure 5. Soluble guanylate cyclase in the KCs is required to form NO-dependent memory.

(A) Diagram of soluble or receptor guanylyl cyclases in Drosophila. (B) RNA-seq data indicate coexpression of Gycα99B and Gycβ100B in KCs, MBONs and DANs. For comparison, the expression levels of other guanylate cyclase genes are also shown. Note that RNA-Seq detected transcripts of neuropeptide gene Nplp1 in both PPL1-γ1pedc and PAM-γ5 (Figure 3—figure supplement 8), but expression of its receptor Gyc76C was barely detectable compared to Gycα99B and Gycβ100B. (C) Induction of Gycβ100B-shRNA in Kenyon cells by activating MB247-switch driver (Mao et al., 2004) with RU-486 feeding reduced the positive-valence memory induced by PPL1-γ1pedc. We also observed a partial effect in the flies without RU-486, presumably due to leaky expression (Figure 5—figure supplement 1E and F). Negative-valence memory with additional feeding of L-DOPA and carbidopa was not affected by Gycβ100B-shRNA induction in KCs. Memories immediately after 3 × 1 min training are shown. The bottom and top of each box represents the first and third quartile, and the horizontal line dividing the box is the median. (D) Induction of scrib-shRNA in KCs also reduced the positive-valence memory induced by activation of PPL1-γ1pedc in a TH mutant background. The whiskers represent the minimum and maximum. N = 12–16. Asterisk indicates significance of designated pair: *, p<0.05; **,p<0.01; n.s., not significant.

Figure 5—figure supplement 1. Expression of Gycbeta100B in the mushroom body lobes (A) Distribution of Gycbeta100B-EGFP in flies carrying the Gycbeta100B[MI08892-GFSTF.2] construct in the MB lobes is shown in a series of anterior to posterior confocal sections.

Right panels show images of wild-type fly, which did not contain the Gycbeta100B-EGFP insertion, prepared with the identical immunolabelling procedure. (B–D) Gycbeta100B-EGFP signals in the γ1 (B) were markedly reduced with induction of Gycβ100B-shRNA HMJ22589 (C) or another RNAi line KK100706 (Dietzl et al., 2007) (D) in Kenyon cells. (E–F) We detected enhanced induction of reporters by MB-switch after feeding RU486 with low but significant basal expression without RU486 feeding.

Figure 5—figure supplement 2. NO from PPL1-γ1pedc activates MBON-γ1pedc but not PAM-DANs in γ3, γ4 and γ5.

(A) In an ex vivo preparation, optogenetic activation of PPL1-γ1pedc evoked slow and sustained calcium response in MBON-γ1pedc. This response was observed in both TH mutant and wild-type background, but diminished in flies fed with L-NNA. N = 6–10. Error bars indicate SEM. Asterisk indicates significance of designated pair: *, p<0.05. (B) Optogenetic activation of PPL1-γ1pedc evoked no obvious calcium response in terminals of PAM-DANs in γ3, γ4 and γ5 compartments.

Figure 5—figure supplement 3. NO is not involved in timing-dependent inversion of valence.

(A) Activation of PPL1-γ1pedc 30 s prior to odor onset induced appetitive memory (known as ‘relief learning’) in a TH wild type background as shown in this panel reproduced from Aso and Rubin (2016). These data were obtained with 1x training whereas a 3x training protocol was used for the experiments shown in (B). N = 10–12. (B) Relief learning was not affected by NOS-knockdown in PPL1-γ1pedc. N = 10. (C–D) Relief learning was completely abolished in a TH mutant background and could be restored with TH expression in PPL1-DANs. N = 12. (E) Paring optogenetic activation of PPL1-γ1pedc with either the first or second presented odor gave similar levels of valence-inverted memory. N = 12. Error bars indicate SEM. Asterisk indicates significance of designated pair in B, D, E or zero in C: *, p<0.001; n.s., not significant.