DOI: 10.5061/dryad.15dv41p39
title: BBB permeability, sleep and qRT-PCR data for: The Drosophila blood-brain barrier regulates sleep via moody GPCR signaling

description: Sleep is vital for most animals, yet its mechanism and function remain
 unclear. We found that permeability of the BBB–the organ required for
 maintenance of homeostatic levels of nutrients, ions, and other molecules
 in the brain–is modulated by sleep deprivation and can cell-autonomously
 effect sleep changes. We observed increased BBB permeability in known
 sleep mutants as well as in acutely sleep deprived animals. In addition to
 molecular tracers, sleep deprivation-induced BBB changes also increased
 penetration of drugs used in the treatment of brain pathologies. After
 chronic/ genetic or acute sleep deprivation, rebound sleep or
 administration of the sleeping aid gaboxadol normalized BBB permeability,
 showing that sleep deprivation effects on the BBB are reversible. Along
 with BBB permeability, RNA levels of the BBB master regulator moody are
 modulated by sleep. Conversely, altering BBB permeability alone through
 glia-specific modulation of moody, gαo, loco, lachesin, or neuroglian –
 each a well-studied regulator of BBB function – was sufficient to induce
 robust sleep phenotypes. These studies demonstrate a tight link between
 BBB permeability and sleep and indicate a novel role for the BBB in the
 regulation of sleep. 
description: Fly genetics Flies were raised on standard
 cornmeal/molasses food at 25°C in a 12-hour light/12-hour dark (LD) cycle.
 Wildtype strains used as controls were isogenic <em>w1118</em>
 (<em>iso1CJ</em>) strains (107,
 108) for sleep. ln
 all experiments, <em>Gal4</em> and <em>UAS</em>
 parental controls were tested as hemizygotes. The following fly stocks
 were used: <em>redeye (rye, Bloomington
 #80692)</em> or <em>nicotinic Acetylcholine Receptor
 α4</em> (61),
 <em>shaker<sup>5</sup> (sh, Bloomington
 #111)</em>, <em>hyperkinetic<sup>2</sup> (hk,
 Bloomington #55)</em>, <em>fumin (fmn, </em>was a lab
 stock<em>)</em>, <em>sleepless<sup>P1</sup>(sss, </em>gift from A. Sehgal<em>),</em>   <em>insomniac<sup>1</sup> (inc)</em>, and <em>wide awakeD1 (wakeD1) </em>and <em>wide awakeD2 (wakeD2, </em>gifts from Mark Wu<em>)</em>, <em>moody-Gal4 </em><em>(44)</em>;<em> UAS-loco </em>(Schwabe et al., 2005);<em> </em><em>GaO-RNAi #3</em>, and <em>repo-GeneSwitch</em> were gifts from U. Gaul, <em>TH-Gal4 </em>was a lab stocks; <em>Tdc2-Gal4</em> and <em>UAS-dTRPA1</em> are from the Bloomington stock center (#9313 and #26264, respectively). <em>nrgRNAi</em>, <em>GaO-RNAi#1, #2 and #</em><em>3, lachesin-RNAi, pkaC1-RNAi, moodyRNAi</em> are VDRC #9248, #28201gd, #19124, #28010, #28940, #31277, #36821, respectively. <em>moody-Gal4</em> was used with <em>UAS-dicer</em> (Bloomington #24651) to enhance RNAi efficiency. All genetic sleep mutants were outcrossed 5 generations to the wild-type strain. For <em>repo-GeneSwitch</em> induction, 5–8 day old flies were placed on food containing 5mM RU486 in ethanol (Abcam; ab120356) for 3 days or only on food containing the corresponding volume of the solvent ethanol, followed by BBB and sleep analysis. Males were used for all experiments except <em>tdc2&gt;TRPA1.</em> qRT-PCR 7-day-old flies were frozen in liquid nitrogen at ZT4. Heads were homogenized with TRIzol (Invitrogen) and RNA was extracted with RNeasy Mini Kit (QIAGEN). cDNA was prepared using iScript Reverse Transcription Supermix (BIO-RAD). qRT-PCR was performed with Fast SYBR Green Master Mix (Applied Biosystems) and 7500 Fast Real-Time PCR System (Applied Biosystems). The following primers were used: <em>moody</em> forward: TCCTTCGTCGTCTGCTACTTG, <em>moody</em> reverse: ATTGTGCGGCTGTGGTTGTTG, <em>gapdh</em> forward: CCACTGCCGAGGAGGTCAACTAC, <em>gapdh</em> reverse: ATGCTCAGGGTGATTGCGTATGC BBB injection assay Modified after Bainton et al. 2005 and Li, 2021. CO2-anesthetized adult flies were injected using a MPPI-3 pressure injector (Applied Scientific Instrumentation) with 1 mm borosilicate needles (FHC-Co) containing fluorescent dyes under a dissecting microscope. An average volume of 100 ± 25 nL dye per injection (range 70–130 nL) of dye was injected into the lateral thorax between wing socket and haltere. Moderately varying dye concentration does not affect brain penetration: up to 4-fold changes in Texas Red 10kDa dye concentration do not significantly affect brain penetration 30 min after injection. All dyes were diluted in injection buffer containing 0.1 mM Sodium Phosphate, pH 6.8 and 5 mM KCl. For all BBB permeability assessments, flies were injected with 2.5 mM Dextran TexasRed (MW 10,000, Thermo Fisher D1863). Fluorescent drugs were injected at the following concentrations: Bocillin FL Penicillin (Thermo Fisher, B13233): 10 mM, Bodipy FL Prazosin (Thermo Fisher, B7433): 15 mM, Vinblastine Bodipy FL (Thermo Fisher V12390): 3 mM, Vancomycin Bodipy FL (Thermo Fisher, V34850): 5 mM. All fluorescent drug derivatives retain bioactivity. Flies were allowed to recover for precisely 30 min before decapitation and imaging on a Zeiss LSM710 confocal microscope. For each fly, both eyes were imaged as interindividual differences in BBB permeability are not larger than intraindividual differences (F-test). Laser and acquisition settings remained unchanged for all samples in the same experiment. Stacks of 6–20 confocal slices of 16 µm were taken and maximum projection images were generated using custom Metamorph (Molecular Devices) script (gift from T. Tong, Bioimaging Resource Center, Rockefeller University). Average pixel intensity in the eye was measured using ImageJ software. For each experiment, data were normalized to mock injected flies. For individual assessment of sleep and BBB permeability, <em>inc</em> flies were loaded into <em>Drosophila</em> activity monitors (Trikinetics) and sleep was recorded for 4 days before injection and BBB assessment. All injections were conducted at ZT0.5-1.5 unless otherwise noted. Sleep analysis 5–7-day-old animals eclosing from light:dark cycle- entrained cultures were loaded into glass tubes and assayed for 5–7 days at 25°C in light:dark cycles. Locomotor activity levels were monitored using the <em>Drosophila </em>Activity Monitoring System (Trikinetics). For sleep measurements, activity counts were collected in 1-minute bins for at least 4 days in light:dark cycles and sleep was identified as at least 5 minutes of inactivity using a sliding window. Sleep parameters were determined using an R-script.  Sleep deprivation and gaboxadol administration Flies were mechanically stimulated overnight during their nighttime sleep hours (zeitgeber time 12–24 h) using a custom-built machine. Mechanical stimuli were applied for 2 seconds at random intervals averaging 20 seconds. Gaboxadol administration was performed as previously described. Briefly, flies were placed into glass tubes with food containing either water or 1 mg/ml of gaboxadol (or THIP) hypnotic. Tubes were loaded into DAM monitors (Trikinetics) and sleep was monitored for 15 h prior to BBB assessment via dye injection. Thermogenetic sleep deprivation was induced by moving <em>TH&gt;TRPA1</em> or <em>Tdc2&gt;TRPA1</em> crosses from 18°C to 29°C for different lengths of time, and back to 18°C for recovery sleep. 
description: # BBB permeability, sleep and qRT-PCR data for "The *Drosophila*
 blood-brain barrier regulates sleep via *moody* G-protein coupled receptor
 signaling"
 [https://doi.org/10.5061/dryad.15dv41p39](https://doi.org/10.5061/dryad.15dv41p39) Shown are: For Figures 1D, E, H, I, K, L, 2B, D, F, 3B, C, F, G, 4C, D, H, I, S1C,D, S2C, S3D, E, F, G: 12 or 24 hour sleep and blood-brain barrier (BBB) permeability data for individual flies. For Figure 3I: Individual quantitative real-time polymerase chain reaction (qRT-PCR) results from 3 separate experiments ## Description of the data and file structure Tab 1: Sleep and BBB data for Figs 1,2,3: Thermogenetic sleep deprivation, mechanical sleep deprivation, drug penetration into the brain Tab 2: Sleep and BBB permeability data for sleep mutants in Figure 3. Tab 3: qRT-PCR data for *moody* RNA in wt, sleep deprivation (SD) and different sleep mutants Tab 4: BBB permeability and sleep parameter data for Supplemental figures BBB permeability units are [relative fluorescence] daily sleep units are [minutes] qRT-PCR data units are [relative expression] ##