BBB permeability, sleep and qRT-PCR data for: The Drosophila blood-brain barrier regulates sleep via moody GPCR signaling
Data files
Sep 27, 2023 version files 331.97 KB
-
Fluorescence__sleep__qRTPCR_data.xlsx
-
README.md
Abstract
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.
README: 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
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]
Methods
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 w1118 (iso1CJ) strains (107, 108) for sleep. ln all experiments, Gal4 and UAS parental controls were tested as hemizygotes. The following fly stocks were used: redeye (rye, Bloomington #80692) or nicotinic Acetylcholine Receptor α4 (61), shaker5 (sh, Bloomington #111), hyperkinetic2 (hk, Bloomington #55), fumin (fmn, was a lab stock), sleeplessP1(sss, gift from A. Sehgal), insomniac1 (inc), and wide awakeD1 (wakeD1) and wide awakeD2 (wakeD2, gifts from Mark Wu), moody-Gal4 (44); UAS-loco (Schwabe et al., 2005); GaO-RNAi #3, and repo-GeneSwitch were gifts from U. Gaul, TH-Gal4 was a lab stocks; Tdc2-Gal4 and UAS-dTRPA1 are from the Bloomington stock center (#9313 and #26264, respectively). nrgRNAi, GaO-RNAi#1, #2 and #3, lachesin-RNAi, pkaC1-RNAi, moodyRNAi are VDRC #9248, #28201gd, #19124, #28010, #28940, #31277, #36821, respectively. moody-Gal4 was used with UAS-dicer (Bloomington #24651) to enhance RNAi efficiency. All genetic sleep mutants were outcrossed 5 generations to the wild-type strain. For repo-GeneSwitch 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 tdc2>TRPA1.
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: moody forward: TCCTTCGTCGTCTGCTACTTG, moody reverse: ATTGTGCGGCTGTGGTTGTTG, gapdh forward: CCACTGCCGAGGAGGTCAACTAC, gapdh 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, inc flies were loaded into Drosophila 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 Drosophila 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 TH>TRPA1 or Tdc2>TRPA1 crosses from 18°C to 29°C for different lengths of time, and back to 18°C for recovery sleep.