Figure 1. A circadian clock in the Drosophila BBB regulates RHB permeability of the brain.

(a–b) Rhythm of permeability into the Drosophila brain: Flies were injected with RHB under CO₂ anesthesia at different time points and the levels of RHB in individual fly brains and bodies were measured after 1 hr using a fluorescence reader at Ex540/Em595. The level of RHB fluorescence in the brain was normalized to the amount of RHB in the body to control for injection consistency. Shown are the brain:body ratios of RHB in a) iso31 n=14–21 per time point, pooled from 3+ experiments and b) per⁰¹ flies. n=8–21, pooled from 2 experiments. Means ± SEM are shown. c) Expression of the PER clock protein in the BBB. PG-GAL4>UAS-nGFP Drosophila were entrained to a 12:12 LD cycle for 3 days. The brain was analyzed for nGFP and PER expression at ZT2, ZT8, ZT14, and ZT20 with a confocal microscope. Representative images of the surface of the brain are shown. Each panel represents an area 100×120μm. (d–e) The PG clock is required for the permeability rhythm. UAS-dncyc (control) and d) PG-GAL4>UAS-dncyc (experimental) or e) UAS-dncyc (control) and SPG-GAL4> UAS-dncyc (experimental) were injected with RHB under CO₂ anesthesia and assessed by Ex540/Em595 fluorescence after 60 mins. Means ± SEM of the ratio of brain:body fluorescence are depicted. n=12–22, pooled from 4 experiments. pCycle indicates a presence of rhythm using JTK cycle analysis. See also Figure S1.