1. Age-Related Changes in Insulin-like Signaling Lead to Intermediate-Term Memory Impairment in Drosophila 
Kento Tanabe, Motoyuki Itoh, Ayako Tonoki 
Cell Reports 
Volume 18, Issue 7, Pages (February 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 1598-1605DOI: (10.1016/j.celrep.2017.01.053)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
IPCs Are Required for Memory Maintenance, but Not Memory Acquisition
(A) The experimental timeline shows the timing of RU486 feedings and memory tests in 10-day-old flies (young, top) or 30-day-old flies (aged, bottom).
(B) The performance index (PI) was significantly reduced 9 hr after training in 10-day-old flies with cell death induced in IPCs (RU+) compared with control flies (RU−) but was not significantly different 3 min or 1 hr after training (∗p < 0.05, n ≥ 6). Fly genotypes: Dilp2-GeneSwitch/+; UAS-reaper/+.
(C) The 3-hr PI was significantly reduced in 10-day-old flies with cell death induced in IPCs (RU+) compared with controls (RU−) (∗∗p < 0.001, n ≥ 12). While the PI was significantly impaired in 30-day-old flies compared to 10-day-old flies (∗p < 0.05), induction of cell death in IPCs was not significantly impaired in 30-day-old flies (p = 0.98, n = 13). Fly genotypes: Dilp2-GeneSwitch/+; UAS-reaper/+.
(D) 10- and 30-day-old flies with cell death induced in IPCs (RU+) and controls (RU−) exhibit similar odor avoidance to both 3-octanol and benzaldehyde (n ≥ 6, n.s., no significant difference).
Results are presented as means ± SEM. See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2 Aging Reduces the Expression of dilp3 in IPCs
(A and B) Aging does not affect the process of IPCs. Projection images of the brains of flies carrying Dilp2-Gal4 and UAS-mCD8::GFP (green) stained with the neuropil marker nc82 (magenta) at 10 or 30 days of age. Scale bar, 50 μm.
(C and D) The number of IPCs did not change with aging. Projection images of the brains of flies carrying Dilp2-Gal4 and UAS-nlsGFP (green) stained with the neuropil marker nc82 (magenta) at 10 or 30 days of age. Scale bar, 20 μm.
(E–H) Aged flies showed reduced expression of dilp3 (F), but not dilp2 (E), dilp5 (G), and inR (H) (∗p < ). qPCR measures of the relative expression levels of each gene in wild-type Canton-S flies at 30 days of age compared 10 days of age. The normalized expression level is shown; n = 3 each.
(I–S) Aged flies showed reduced protein levels of Dilp3, but not Dilp2. Projection images of the brains of flies carrying Dilp2-Gal4 and UAS-mCD8::GFP (green) stained with Dilp2 (magenta) and Dilp3 (blue) at 10 days of age (I–L) and 30 days of age (M–P). Scale bar, 20 μm. The mean fluorescence intensity in the cell body of IPCs is shown (Q–S). Data were assessed by t test and are presented as means ± SD; n ≥ 9. p < a.u., arbitrary units.
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5. Figure 3
The Transient Expression of dilp3 Is Required for Memory Maintenance, but Not Memory Acquisition
(A) The PI measured immediately after conditioning was indistinguishable between 10-day-old flies expressing dilp3RNAi-JF01345 (dilp3RNAi-A) in IPCs (black bar) and controls (white or gray bars) (n ≥ 7).
(B) The 3-hr PI measured after conditioning was significantly reduced in flies expressing dilp3RNAi-A in IPCs (black bar) compared to controls (white or gray bars) (∗p < 0.005; n ≥ 6).
(C) The PI was significantly reduced 3 hr after conditioning in homozygous dilp3 mutants compared to heterozygous dilp3 mutants (p < 0.05, n ≥ 8), but not change was observed immediately after conditioning (n = 7).
(D) The 3-hr PI measured after conditioning was not significantly altered in flies expressing dilp2RNAi-HMS00476 (dilp2RNAi) or dilp5RNAi-HMS00548 (dilp5RNAi) in IPCs (black bar) compared to controls (white or gray bars) (n ≥ 8).
(E) The 3-hr PI was significantly reduced in flies transiently expressing dilp3RNAi-JF01345 (dilp3RNAi-A) or dilp3RNAi-HMS00546 (dilp3RNAi-B) in IPCs (red bar), but not in the control group (blue bar) (∗∗p < , n ≥ 8), using TARGET system. Flies were reared through development at 18°C until 5 days of age, when they were shifted to 30°C maintenance for 5 days, at which point the memory assay was performed. Fly genotypes: Dilp2-Gal4/UAS-dilp3RNAi-A (or -B); tub-Gal80ts/+, Dilp2-Gal4/+; tub-Gal80ts/+.
Results are presented as means ± SEM. See also Figure S2.
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6. Figure 4
Insulin Receptor Expression in Fat Bodies Is Critical for Memory Maintenance
(A and A′) The PI was significantly reduced at 3 hr after conditioning in flies expressing dominant-negative InR in the whole body (RU+) compared to controls (RU−) (p < 0.05; n ≥ 13) (Aʹ) but was not different immediately after conditioning (p = 0.83; n ≥ 5) (A). Fly genotype: Tubulin-GeneSwitch/UAS-InR-DN.
(B and B′) The PI was not significantly reduced immediately after conditioning (p = 0.17; n = 12) (B) or 3 hr after conditioning in flies expressing dominant-negative InR in all neurons (RU+) compared to controls (RU−) (p = 0.53; n ≥ 11) (Bʹ). Fly genotype: Elav-GeneSwitch/UAS-InR-DN.
(C and C′) The PI was not significantly reduced immediately after conditioning (p = 0.82, n = 6) (C) or 3 hr after conditioning in flies expressing dominant-negative InR in the mushroom body neurons (RU+) compared to the control group (RU-) (p = 0.75, n = 6) (C′). Fly genotype: 12-1 (MB-GeneSwitch)/UAS-InR-DN.
(D and D′) The PI was not significantly reduced immediately after conditioning (p = 0.26; n ≥ 5) (D) or 3 hr after conditioning in flies expressing dominant-negative InR in glia (RU+) compared to controls (RU−) (p = 0.40; n ≥ 9) (D′). Fly genotype: Glia-GeneSwitch /UAS-InR-DN.
(E and E′) The PI was significantly reduced 3 hr after conditioning in flies expressing dominant-negative InR in the FB (RU+) compared to controls (RU−) (∗p < 0.05; n ≥ 8) but was not different immediately after conditioning (p = 0.98; n ≥ 8). Fly genotype: FatBody-GeneSwitch1.32/UAS-InR-DN.
(F) The PI was significantly reduced 3 hr after conditioning in flies expressing dominant-negative InR in the FB (31°C) compared to controls (18°C) as assessed using the TARGET system (∗p < 0.01; n ≥ 6). Fly genotype: tub-Gal80ts/+; Cg-Gal4/UAS-InR-DN.
(G and H) The 3-hr PI was significantly increased in flies with dilp3 induced in IPCs (RU+) compared with controls (RU−) at both 10 days of age (G) (∗p < 0.05; n = 8) and 30 days of age (H) (∗p < 0.05; n ≥ 11). The experimental timeline above the graph shows the times of RU486 feedings and memory tests in 10-day-old flies (G) or 30-day-old flies (H). Fly genotype: Dilp2-GeneSwitch/+, Dilp2-GeneSwitch/+; UAS-dilp3/+.
(I) Model illustrating the role of insulin-like signaling in memory maintenance and age-related memory impairment. Memory maintenance requires transient systemic IIS for inter-organ communication between IPCs and the FB. Age-related decline in IIS and the downregulation of dilp3 contribute to age-related deficits in memory maintenance.
Results are presented as means ± SEM. See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
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