1. Visual Working Memory Requires Permissive and Instructive NO/cGMP Signaling at Presynapses in the Drosophila Central Brain 
Sara Kuntz, Burkhard Poeck, Roland Strauss 
Current Biology 
Volume 27, Issue 5, Pages (March 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 613-623DOI: (10.1016/j.cub.2016.12.056)
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3. Figure 1 NOS Expression in the Adult Brain
(A) Total z projection of an adult wild-type (WT) brain with antibody labeling against NOS. The ellipsoid body (EB), the subesophageal ganglion (SOG), and some cell bodies (CB) are indicated.
(B) Co-labeling of NOS (magenta) and Fasciclin II (FASII; green). Co-localization is seen in the EB (24 μm z projection), but not in the α lobes or peduncle (PD) of the mushroom bodies. Intermittent layers of the fan-shaped body (FB; 36.75 μm z projection) show elevated NOS expression.
(C–F) Co-localization analysis of NOS expression in R1 (C), R2 (D), R3 (E), and R4 (F) neurons of the EB. The different R subtypes are labeled by the expression of UAS-CD4-tdGFP using indicated GAL4 drivers. 2 μm optical sections with the strongest GFP expression of each R neuron driver line and the corresponding NOS staining are shown. BU, bulb.
(G–J) Merge of NOS and GFP expression in cell bodies of the analyzed R1 (G), R2 (H), R3 (I), and R4 (J) neurons; optical sections 0.75 μm.
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4. Figure 2 NOS Is Required for the Orientation Memory
(A) In the detour paradigm, a fly heading toward a vertical stripe is distracted at the midline by switching the stripe to + or −90°. The distractor stripe also disappears 1 s after the fly has turned toward it; no visual cues are left.
(B) In comparison to wild-type CS, homozygous NosΔ15 and transheterozygous NosΔ15/NosC or Nose2671/NosΔ15 flies possess no or reduced memory (n = 17–18; Kruskal-Wallis analysis; Bonferroni post hoc).
(C and D) Reduced NOS expression in R3 neurons through RNAi-mediated knockdown (189Y-GAL4; UAS-NosRNAi) is detectable in the EB (D) in comparison to UAS-NosRNAi controls (C; 3.75 μM z projection; for specificity, see Figure S1). Boxed areas were used for quantification of the NOS signal of the EB (C and D) in relation to the FB (C’ and D’; mean pixel intensities: NosRNAi/+: EB/FB = 4.57 ± 0.22; 189Y>NosRNAi: EB/FB = 2.39 ± 0.35; n = 5; mean ± SEM; t test; p < 0.001).
(E) Nos-RNAi induction in R3 neurons impaired the working memory (n = 20; Mann-Whitney U test; for all statistics, see Table S1).
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5. Figure 3 Elevated NO Signaling Rescues the ebo Mutant Phenotype
(A) Leaky expression from the UAS-Nos transgene elevates the memory score, which is not improved by targeted expression in R3/R4d via c232-GAL4 (n = 18; Kruskal-Wallis analysis; Bonferroni post hoc).
(B and C) The VT5535-GAL4 driver line expresses tdGFP in NOS-positive neurons innervating the dorsal fan-shaped body (FB; C), but not the ellipsoid body (EB; 0.75 μM z projection; B).
(D) RNAi-mediated knockdown of Nos in these FB neurons reduces the orientation memory (n = 20; 15 Mann-Whitney U test); for statistics, see Table S2.
(E) Overexpression of Nos in R3 or R2 neurons restores the ebo678 memory deficit (n = 24; Kruskal-Wallis analysis; Bonferroni post hoc; for all statistics, see Table S2).
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6. Figure 4 CBS Is Required for the Orientation Memory
(A) CbsEY04457 mutants exhibit an impaired memory relative to wild-type CS flies that was rescued by expression of CBS (UAS-Cbs) in R2 and R3 neurons, but not by expression in the olfactory receptor cells (Or83b-GAL4; n = 18–20; Kruskal-Wallis analysis; Bonferroni post hoc).
(B) Expression of CBS in R2 or R3 neurons in the ebo678 mutant rescued the memory deficit in comparison to CS flies (n = 24; Kruskal-Wallis analysis; Bonferroni post hoc; related western blot analysis in Figure S2).
(C) 24 hr of cysteine supplementation (20 mM N-acetyl-L-cysteine) to the medium rescues the memory performance of ebo678 mutant flies (n = 15 each group; Wilcoxon matched-pairs test; for all statistics, see Table S3).
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7. Figure 5 NO and H2S Induce Elevated cGMP Levels
(A) Flies double heterozygous for NosC and a deficiency for the soluble guanylyl cyclase (cGC; Df(3R)BSC547) reveal a complete loss of orientation memory in comparison to the heterozygous controls (n = 15; Kruskal-Wallis analysis; Bonferroni post hoc).
(B) Heterozygous controls for the NosC- and for02-null alleles behave wild-type like, whereas double heterozygous flies lack this working memory (n = 15; Kruskal-Wallis analysis; Bonferroni post hoc; related western blot analysis in Figure S2).
(C) Heterozygosity for the phosphodiesterase 6 (Pde6MI04678) suppressed the memory deficit of the CbsEY04457 mutant (n = 19–20; Kruskal-Wallis analysis; Bonferroni post hoc).
(D) ebo mutants show a defective orientation memory, whereas heterozygous Pde6 flies behave wild-type like. Crossing a mutant Pde6 allele into a hemizygous ebo678 background completely rescues the ebo mutant phenotype (n = 20; Kruskal-Wallis analysis; Bonferroni post hoc).
(E) Knocking down PDE6 by driving Pde6-RNAi in R3 neurons (189Y-GAL4) in an ebo678 mutant background partially restores the memory deficits of the mutant (n = 20; Mann-Whitney U test).
(F) Feeding the PDE6 inhibitor Zaprinast (100 μM) restores the memory deficits of ebo678, CbsEY04457, and NosΔ15 mutants. The same individuals were tested before and after 24 hr treatment (n = 14–15; Wilcoxon matched-pairs test).
(G) Knocking down the soluble guanylyl cyclase GCα99B in R3 neurons (189Y-GAL4) reduces the orientation memory (n = 18; Mann-Whitney U test; for all statistics, see Table S4).
(H) Schematic drawing of the postulated pathway.
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8. Figure 6 NO and H2S Signaling Converge in dCREB Activation
(A) Inducing a dominant-negative form of dCREB in adult R3 neurons (189Y Tub>GAL80ts>UAS-dCreb2-b) resulted in a complete loss of orientation memory. The same flies were tested before (18°C) and 24 hr after (30°C) induction of GAL4 activity (n = 16; paired t test).
(B) Heterozygous CrebBS162 mutants show a defective memory in comparison to wild-type CS. Driving the CrebB cDNA in R3, but not in R1/R2/R4 neurons, restores the memory (n = 20; Kruskal-Wallis analysis; Bonferroni post hoc).
(C) Overexpressing CrebB in R3 neurons of ign58/1 mutants rescues the memory deficit (n = 15; Kruskal-Wallis analysis; Bonferroni post hoc).
(D) Western blot analysis (ten heads per lane) using Drosophila-specific antibodies for phosphorylated dCREB (P∼dCREB) and dCREB revealed a 10% reduction of P∼dCREB in ign58/1 mutants in comparison to wild-type CS. Intensities were measured in the area indicated by the curly brace and normalized for each genotype (mean and SEM values; n = 9; t test; p = 0.013).
(E) Transheterozygous homerR102/homerEP2141 flies possess no orientation memory, but expression of homer in R3 rescues to wild-type levels (n = 18; Kruskal-Wallis analysis; Bonferroni post hoc).
(F) 189Y Tub>GAL80ts>UAS-homerRNAi flies reared at 18°C displayed a normal memory score. After 24 hr incubation at 30°C to induce the RNAi knockdown, the same flies have lost orientation memory (n = 14; paired t test; for all statistics, see Table S5).
(G) Schematic of the NO/H2S signaling pathway for the orientation memory.
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9. Figure 7
Regional NO Signaling in the EB Is the Substrate of the Memory Trace
(A) Feeding 100 μM NOS inhibitor L-NNA for 5 min immediately reduces the visual memory close to chance levels (n = 16); 10 μM L-NNA reduced the memory to a lesser extent (n = 15); Wilcoxon matched-pairs test.
(B) Inducing RNAi against cyclic nucleotide-gated ion channel subunits CngA or Cngl for 48 hr in adult R3 neurons reduced the memory in comparison to un-induced controls (n = 15 each; t test for dependent samples).
(C) Inducing overexpression of two cDNA copies of NOS (overnight 30°C) in R3 neurons eliminates the orientation memory in comparison to matched un-induced control flies (n = 12; Wilcoxon matched-pairs test; for all statistics, see Table S6).
(D) Schematic drawing of the proposed model depicting connectivity and flux of information in one wedge/segment of the ellipsoid body (EB) and between the EB and the protocerebral bridge (PB). R3 (black) receives visual input from R2/R4 (green) and simultaneously from an idiothetic neuron (blue) conveying information about the fly’s moves with respect to its surrounding. This synergistic activation stimulates NO production in one axonal branch of an R3 neuron, and elevated levels of NO induce cGMP. cGMP-mediated opening of cyclic nucleotide-gated ion channels (CNGs; violet) result in Ca2+ influx, representing the memory trace of the landmark. When no object is visible (off signal), a columnar neuron (red) projecting from the EB to the PB receives activation through the R3 neurons. Differential activation of the EB-PB neurons in the wedges is then used to steer the fly.
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