Extended Data Fig. 2. Central brain lipid droplets in Drosophila are localized primarily to glia and increase with neural activity.
(A) The lipophilic dyes, BODIPY 493 and Nile Red, colocalize in Drosophila brains and can be used interchangeably. Here, the anterior region of a single, central brain is shown stained with both dyes. (A, right- green) BODIPY 493 staining, (A, center -magenta) Nile Red staining and (A, left) merge of both channels with colocalization in white. (n = 6 brains were imaged to verify colocalization between BODIPY and Nile Red.) (B and C) The lipid droplet marker, UAS-LD-GFP, was expressed in glia with Repo-GAL4 or in neurons with Nsyb-GAL4 and stained with the lipophilic dye, Nile Red. (B) LD-GFP expressed in glia was found to localize in a ring around nearly all Nile-Red stained lipid droplets (n = 7/7 of brains imaged showed GFP and Nile Red colocalization). (C) Conversely, neuronally-expressed LD-GFP exhibited a very bright, diffuse localization pattern in neuropil and the periphery of cell bodies and was never seen in a ring pattern around any lipid droplets stained by Nile Red (n = 0/7 of brains imaged showed GFP and Nile Red colocalization). For all images, the central brain is shown on the left with a white box indicating the enlarged region shown on the right. Arrows mark the locations of representative lipid droplets. Scale bars at left are 50uM each and at right (enlarged) are 10uM each. All images were acquired with sequential, independent imaging of red and green excitation/emission. Proper dye-staining protocols can be found in the Methods section and were critical for visualization of central brain lipid droplets. (D) Known patterns of time spent awake (yellow) and time spent asleep (blue) throughout the day in Drosophila on a 12:12 light: dark cycle or following sleep deprivation (dotted). (E) Schematic summarizing experimental timepoints from all figures, connected by lines, from lipid droplet count (dark blue) and glial mito-roGFP2-Grx1 oxidation (red) experiments showing when each metric is or is not consistently increased. While both dependent on prior wake, increases in mito-roGFP2-Grx1 oxidation are most consistent during known wake periods (ZT0 + SD and ZT12, as in D), while lipid droplet count only increases consistently during periods with some sleep (ZT2 + SD, ZT6, ZT14, ZT18, as in D). In flies on RU-486, mito-roGFP-Grx1 is most consistently increased after SD and lipid droplets are most consistently increased at ZT18. (F) Activity counts per 30 minutes (left) and sleep per 30 minutes (right) of flies with dTrpA1-induced neuronal hyperactivation (red) and controls (grey). The temperature was increased from 23 °C to 25.5 °C at ZT0 and remained at 25.5°C from ZT0-ZT9. (G) Hyperactivation of all neurons with Nsyb>dTrpA1 (25.5 °C) from ZT0-ZT9 causes a further increase in lipid droplet accumulation over controls. For all data shown, *=p < 0.05, **=p < 0.01, ***=p < 0.001 and ****=p < 0.0001. Bars/ error bars indicate mean and SEM, respectively. Data points indicate individual flies/brains. Number of flies (n) as plotted from left to right and statistical test used: (F-G) n = 22,23,23; Mann-Whitney-two tailed.