AANAT1 functions in astrocytes to regulate sleep homeostasis
- Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, McGill University, Canada
- BRaIN Program, Research Institute of the McGill University Health Centre, Canada
- Integrated Program in Neuroscience, McGill University, Canada
- Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, United States
- Biology Graduate Group, University of Pennsylvania, United States
- Howard Hughes Medical Institute, Chronobiology and Sleep Institute, Perelman School of Medicine, University of Pennsylvania, United States
Figures
Figure 1 with 1 supplement
AANAT1 expression in the adult Drosophila brain.
(A–B) AANAT1 IHC in the ventral nerve cords of age-matched embryos (stage 17) of a w1118 control (A) and an AANAT1-null homozygous for Df(BSC)356 (B). (C–M) AANAT1 IHC in the central brain of …
Figure 1—figure supplement 1
AANAT1 expression in the adult Drosophila brain.
(A) Single optical slice showing AANAT1 (magenta), Elav (blue) and Alrm >nuRFP (green), with AANAT1-negative astrocytes (white arrowheads) in the optic lobe. (B) Single optical slice showing AANAT1 …
Figure 2 with 1 supplement
Characterization of AANAT1lo.
(A) Western blot of lysates prepared from dissected brains (ZT9-10) of iso31, AANAT1lo and AANAT1lo/Df(In(2LR)Px[4]) adult males. (B) Quantification of AANAT1 expression normalized to that of actin …
Figure 2—figure supplement 1
Characterization of AANAT1lo.
(A) Quantification of Ebony expression normalized to that of actin (mean + standard deviation, n = 3 biological replicates, one-way ANOVA with Tuckey’s post-hoc test). (B) HPLC-MS measurement of …
Figure 3 with 1 supplement
AANAT1 knockdown in neurons.
(A–D) Baseline sleep upon knockdown with UAS-HMS01617 (RNAi 1). 24 hr sleep profile showing light/dark conditions on X-axis (A), and quantification during day (ZT 0–12) versus night (ZT 12–24) of …
Figure 3—figure supplement 1
AANAT1 knockdown in neurons.
(A) Activity Index for knockdown with UAS-HMS01617 (RNAi 1) for the nSyb-Gal4 control (black; n = 30), the UAS-HMS01617 control (blue; n = 32), and knockdown animals (nSyb >HMS01617, green; n = 32). …
Figure 4 with 1 supplement
AANAT1 knockdown in astrocytes.
(A) Compared with Alrm-Gal4 controls (Alrm>), the number of nuRFP labeled astrocytes in the central brain is unaffected upon RNAi knockdown of AANAT1 with HMS01617 (AANAT1-RNAi 1) or JF02142 …
Figure 4—figure supplement 1
AANAT1 knockdown in astrocytes.
(A) Activity Index for knockdown with UAS-HMS01617 (RNAi 1) for the Alrm-Gal4 control (black; n = 32), the UAS-HMS01617 control (blue; n = 26), and knockdown animals (Alrm >HMS01617, green; n = 32). …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | AANAT1 | Flybase | FBgn0019643 | Previously known as Dat. Also, speck (sp) |
| Genetic reagent (D. melanogaster) | Trh-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_52249 | FlyBase genotype: y1 w*; wgSp-1/CyO, P{Dfd-EYFP}2; P{Trh-GAL4.S}attP2 |
| Genetic reagent (D. melanogaster) | TH-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_8848 | FlyBase genotype: w*; P{ple-GAL4.F}3 |
| Genetic reagent (D. melanogaster) | Tdc2-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_52243 | FlyBase genotype: y1 w*; wgSp-1/CyO, P{Dfd-EYFP}2; P{Tdc2-GAL4.S}attP2 |
| Genetic reagent (D. melanogaster) | 10X-UAS-mCD8-GFP | Bloomington Drosophila Stock Center | RRID:BDSC_32186 | FlyBase genotype: w*; P{10XUAS-IVS-mCD8::GFP}attP40 |
| Genetic reagent (D. melanogaster) | UAS-RFP.nls | Bloomington Drosophila Stock Center | RRID:BDSC_30558 | FlyBase genotype: w[1118]; P{w[+mC]=GAL4-Act5C(FRT.CD2).P}S, P{w[+mC]=UAS RFP.W}3/TM3, Sb[1] |
| Genetic reagent (D. melanogaster) | Mi{MIC} VGlutMI04979 | Bloomington Drosophila Stock Center | RRID:BDSC_38078 | FlyBase genotype: y1w*; Mi{MIC}VGlutMI04979 |
| Genetic reagent (D. melanogaster) | Gad1-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_51630 | FlyBase genotype: P{Gad1-GAL4.3.098}2/CyO |
| Genetic reagent (D. melanogaster) | Cha-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_6793 | FlyBase genotype: w*; P{ChAT-GAL4.7.4}19B P{UAS-GFP.S65T}Myo31DFT2 |
| Genetic reagent (D. melanogaster) | R56F03-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_39157 | FlyBase genotype: w[1118]; P{y[+t7.7] w[+mC]=GMR56 F03-GAL4}attP2 |
| Genetic reagent (D. melanogaster) | AANAT1lo | Bloomington Drosophila Stock Center | RRID:BDSC_3193 | FlyBase genotype: bw[1] AANAT1[lo] |
| Genetic reagent (D. melanogaster) | Df(2R)BSC356 | Bloomington Drosophila Stock Center | RRID:BDSC_24380 | FlyBase genotype: w[1118]; Df(2R)BSC356/SM6a |
| Genetic reagent (D. melanogaster) | In(2LR)Px4 | Bloomington Drosophila Stock Center | RRID:BDSC_1473 | FlyBase genotype: In(2LR)Px[4], dpy[ov1] b[1]/CyO |
| Genetic reagent (D. melanogaster) | tubGal80ts | Bloomington Drosophila Stock Center | RRID:BDSC_7018 | FlyBase genotype: w[*]; sna[Sco]/CyO; P{w[+mC]=tubP-GAL80[ts]}ncd[GAL80ts-7] |
| Genetic reagent (D. melanogaster) | UAS-HMS01617 | Bloomington Drosophila Stock Center | RRID:BDSC_36726 | FlyBase genotype: y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01617}attP40/CyO |
| Genetic reagent (D. melanogaster) | UAS-JF02142 | Bloomington Drosophila Stock Center | RRID:BDSC_26243 | FlyBase genotype: y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02142}attP2 |
| Genetic reagent (D. melanogaster) | MCFO | Bloomington Drosophila Stock Center | RRID:BDSC_64085 | FlyBase genotype: FlpG5.Pest; 10xUAS(FRT-stop)myr::smGdP-HA, 10xUAS(FRT-stop)myr::smGdP-V5-THS-10xUAS(FRT-stop)myr::smGdP-FLAG |
| Genetic reagent (D. melanogaster) | Alrm-Gal4 | Marc Freeman | ||
| Genetic reagent (D. melanogaster) | Eaat1-Gal4 | Marc Freeman | ||
| Genetic reagent (D. melanogaster) | nSyb-Gal4 | Julie Simpson, Stefan Thor | ||
| Genetic reagent (D. melanogaster) | UAS-AANAT1 | This paper | See Materials and methods: Fly stocks | |
| Cell lines (Escherichia coli) | GH12636 | Drosophila Genomic Research Centre | FBcl0129063 | |
| Cell lines (Escherichia coli) | pJFRC-MUH | Addgene | RRID:Addgene_26213 | |
| Antibody | AANAT1 (Rabbit polyclonal) | MEDIMABS, Montreal | This paper | IHC(1:2000), WB (1:2500). See Materials and methods: Generation of AANAT1 antibody |
| Antibody | Elav (Rat monoclonal) | Developmental Studies Hybridoma Bank | Elav-7E8A10 | IHC(1:100) |
| Antibody | Repo (Mouse monoclonal) | Developmental Studies Hybridoma Bank | 8D12 | IHC(1:50) |
| Antibody | Brp (Mouse monoclonal) | Developmental Studies Hybridoma Bank | nc82 | IHC(1:50) |
| Antibody | GFP (Mouse monoclonal) | Clontech | #632381 | IHC(1:200) |
| Antibody | Ebony (Rabbit polyclonal) | Sean Carroll | WB (1:3000) | |
| Antibody | Actin (Mouse monoclonal) | Sigma | #A4700 | WB (1:3000) |
| Commercial assay or kit | HyGLO Chemiluminescent HRP Antibody Detection Reagent | Denville Scientific | ||
| Chemical compound, drug | Formic acid | Fisher scientific | ||
| Software, algorithm | Graphpad Prism 6 | RRID:SCR_002798 | ||
| Software, algorithm | Fiji | RRID:SCR_002285 |
