Fig. 5: DTk neurons and DN1Ps were anatomically and functionally connected.
From: Metabolic control of daily locomotor activity mediated by tachykinin in Drosophila
a, b Fly locomotor activity for w1118 (control) or UAS-DTk Ri (DTk Ri) driven by different DTk-Gal4 (DTk5Fa-Gal4, DTk2Ma-Gal4, and R65E09-Gal4) was analyzed in NSD and HSD under a 16L:8D cycle at 29 °C. a Daily activity profiles of flies on day 7 are shown. b ΔM activity offsets on day 7 are shown. DTk knockdown in DTk5Fa-Gal4, and 65E09-Gal4 active cells abolished the HSD effect, but not in the DTk2Ma-Gal4 active cells. (n = 14–26). Statistically significant differences in ΔM activity between control and DTk knockdown flies (independent t test): *P < 0.05, ***P < 0.001. c, d Flies were entrained in 16L:8D cycle at 23 °C for 7 days in NSD. The temperature was then elevated to 29 °C for 2 more days. c Daily activity profiles of flies on the last day at 23 °C (before activation) and on the 2nd day at 29 °C were overlaid. d M activity offset of individual flies on the last day at 23 °C and on the 2nd day after the temperature elevation to 29 °C to avoid temperature change-induced strong startle activity are shown. Bars indicate mean ± SEM (n = 32). Statistically significant differences in the average time between 23 and 29 °C (independent t test): *P < 0.05. e–l Flies of the indicated genotypes were maintained on a 16L:8D cycle at 29 °C. Brains were dissected at ZT2. e, f On day 7, GRASP-positive signals were produced between DN1ps in R18H11-LexA > LexAop-CD4-spGFP11 and DTk neurons in DTk5Fa > UAS-CD4-spGFP1-10, but not between LNvs in PDF-LexA > LexAop-CD4-spGFP11 and DTk neurons in DTk5Fa > UAS-CD4-spGFP1-10. GRASP signals were detected more broadly in flies fed a HSD. All scale bars represented 20 μm. f The areas showing GRASP signals were quantified using ImageJ software (n = 8–11). Statistically significant differences in GRASP area between NSD and HSD groups (independent t test): ***P < 0.001. g, h GRASP signals between DTk neurons in DTk5Fa > UAS-CD4-spGFP1-10 and DN1ps in R18H11-LexA > LexAop-CD4-spGFP11 were analyzed on days 3, 5, and 7. All scale bars represented 20 μm. h The areas showing GRASP signals were quantified using ImageJ software. The GRASP areas were progressively increased over time in flies fed a HSD. Values indicate mean ± SEM (n = 8–9). Statistically significant differences in GRASP area (one-way ANOVA): ***P 0.001. i–k Flies of the indicated genotypes were maintained on a 16L:8D cycle at 29 °C. Brains were dissected at ZT2. On day 7, nSyb-GRASP-positive signals were produced between DN1ps in R18H11-LexA > LexAop-CD4-spGFP11 and DTk neurons in DTk5Fa > UAS-nSyb-spGFP1-10 (i), but not between DN1ps in R18H11-LexA > LexAop-nSyb-spGFP1-10 and DTk neurons in DTk5Fa > UAS-CD4-spGFP11 (j). Stronger nSyb-GRASP signals were detected when brains were exposed to KCl (final 70 mM, +) than to AHL (−). All GRASP- and nSyb-GRASP-positive signals represented endogenous GFP fluorescence. Brains were counter-stained with anti-NC82 (magenta) antibodies. All scale bars represented 20 μm. k The areas showing nSyb-GRASP-positive signals were quantified using ImageJ software (n = 6–9). Statistically significant differences in nSyb-GRASP area between NSD and HSD groups (independent t test): ***P < 0.001. l Flies of the indicated genotypes (denoted on top) were maintained on a 16L:8D cycle at 29 °C. Brains were dissected at ZT2 and stained with anti-GFP (green), anti-CLK (gray), and anti-NC82 (magenta) antibodies. The right panel shows magnified images of the boxed regions in the left panel. All scale bars represented 20 μm.
