Figures and data in AANAT1 functions in astrocytes to regulate sleep homeostasis

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4 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement

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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 adults. (C) Z-stack projection showing AANAT1 (green) and neuropil marker nc82 (magenta) in Alrm-Gal4/+ control animals. (**D–D'**) Single optical slice showing AANAT1 (magenta) and the pan-neuronal marker Elav (green). Yellow arrowheads point to neurons co-expressing both (D'). (**E–E'**). Single optical slice of AANAT1 (magenta) and the pan-glial marker Repo (green) in control animals where most glia express AANAT1 (E'; yellow arrowheads), but not all (E'; white arrowheads). AL = antennal lobe. Glia were labeled less intensely for AANAT1 than neurons, and so the adjusted imaging parameters reveal higher background levels in the antennal lobe than seen in D-D'. (**F–F''**) Single slice of AANAT1 (magenta), Elav (blue), and astrocyte marker Alrm-Gal4; UAS-nuRFP (Alrm >nuRFP, green) showing co-expression of AANAT1 and nuRFP in astrocytes (F''; yellow arrowheads) and with Elav (F''; yellow asterisk). (**G–G''**) MCFO-labeled single-cell astrocyte clones (anti-V5, green) co-labeled with AANAT1 (magenta). Yellow arrowheads indicate AANAT1-positive astrocyte cell bodies and cytoplasm. AL = antennal lobe. (**H–M''**) Z-stack projections and single=slice images of AANAT1 (magenta) and GFP (green) IHC in monoaminergic neurons labeled with type-specific Gal4 drivers. Dotted boxes in H, J and L show regions approximating those selected for imaging at higher power in animals of the same genotypes shown in I, K and M, respectively. AANAT1 is expressed in some serotonergic neurons (I''; yellow arrowhead), but not in dopaminergic or octopaminergic neurons (K'', M''; white arrowheads). (N) Quantification of the mean number of GFP-positive and GFP/AANAT1 double-positive cells in the central brains of animals where Gal4 is used to express GFP in serotonergic (green), dopaminergic (blue) or octopaminergic (red) neurons. Error bars represent standard deviation. (O) Summary of AANAT1 expression in cell types of the adult *Drosophila* central brain. Scale bars in A, B, D-G, I, K, M = 20 µm. Scale bars in C, H, J, L = 50 μm.

Figure 1—figure supplement 1

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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 (magenta) and R56F03 > mCD8 GFP (green) showing absence of AANAT1 in ensheathing glia (white arrowheads). (**C–F**) AANAT1 expression upon AANAT1 knockdown with UAS-HMS01617 RNAi driven in all glia (C; Repo-Gal4;UAS-Dcr-2), all neurons (D; nSyb-Gal4), astrocytes (E; Alrm-Gal4,UAS-nuRFP), and both in neurons and astrocytes (F; nSyb-Gal4;Alrm-Gal4). Cyan arrowheads in C and E depict axonal bundles. Blue arrowhead in F shows background signal. (**G, H**) Single slices of IHC for AANAT1 (magenta) and Elav (green) showing loss of AANAT1 expression (white arrowheads) upon knockdown in neurons using nSyb-Gal4 to drive either UAS-HMS01617 (G; RNAi 1) or UAS-JF02142 (H; RNAi 2). (**I, J**) Single slices of IHC for AANAT1 (magenta) and Alrm >nuRFP (green) showing loss of AANAT1 expression (white arrowheads) upon knockdown in astrocytes using Alrm-Gal4 to drive RNAi 1 (I) or RNAi 2 (J). Yellow arrowheads indicate AANAT1 expression in neurons. (**K–P**) Z-stack projections showing AANAT1 (gray) expression in ellipsoid body (**K,M,O**) and fan-shaped body (**L,N,P**) neuropil regions in UAS-JF02142/+ controls (**K,L**) and upon knockdown of AANAT1 with AANAT1-RNAi2 driven in astrocytes using Alrm-Gal4 (**M,N**) or in neurons using Elav-Gal4 (**O,P**). (Q) Single-slice of AANAT1 IHC (gray) showing absence of AANAT1 expression in the α and βγ lobes of the mushroom body (outlined with dotted yellow line). (**R–W''**) Z-stack projections (**R,T,V**) and single-slice images (S-S'';U-U'';W-W'') of AANAT1 (magenta) and GFP (green) IHC in non-monoaminergic neurons labeled with type-specific drivers. Dotted boxes in R, T and V show regions approximating those selected for imaging at higher magnification in animals of the same genotypes shown in S-S'', U-U'' and W-W'', respectively. AANAT1 is expressed in subsets of glutamatergic, GABAergic and cholinergic neurons (S'', U'', W''; yellow arrowheads). Scale bars in A, C-F, R, T, V = 50 µm. Scale bars in B, G-J, K-P, S'', U'', W''=20 μm.

Figure 2 with 1 supplement

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Characterization of *AANAT1^lo*.

(A) Western blot of lysates prepared from dissected brains (ZT9-10) of iso31, *AANAT1^lo* and *AANAT1^lo*/Df(In(2LR)Px[4]) adult males. (B) Quantification of AANAT1 expression normalized to that of actin (mean + standard deviation, n = 3 biological replicates). (**C–E**) Z-stack images showing AANAT1 (gray) in iso31 (C), *AANAT1^lo* (D) and *AANAT1^lo*/Df(In(2LR)Px[4]) (E) animals. Blue arrowhead in E represents background signal. Scale bars = 50 µm. (**F, G**) Single optical slices showing AANAT1 (gray or magenta) and Elav (green) in iso31 (**F, F'**) and *AANAT1^lo* (**G, G'**). Yellow arrowhead shows AANAT1⁺ astrocytes. Scale bars = 20 µm. (H) Schematic of experiment for HPLC-MS analysis. (I) HPLC-MS measurement of serotonin (one-way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01,) and dopamine (Kruskal–Wallis test, Dunn’s multiple comparisons, *p<0.05,) in iso31 (black) and *AANAT1^lo* (red) fly brains under control and sleep deprivation (SD) conditions. Box and whisker plots show 25–75% interquartile range (box), minimum and maximum (whiskers), median (horizontal line in box), and mean (+). n = 5 per genotype. (J) Western blot of lysates prepared from dissected brains (ZT24-25) of iso31 females in control (no SD) and SD conditions. (K) Quantification of AANAT1 (paired t-test, p=0.0831, n = 3) expression, normalized to actin levels in iso31 animals under control (no SD) and SD conditions.

Figure 2—figure supplement 1

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Characterization of *AANAT1^lo*.

(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 octopamine (Mann-Whitney test, p<0.533) in *AANAT1^lo* animals under control (n = 2) and SD (n = 5) conditions. Box and whisker plot as in Figure 2F. (C) Quantification of Ebony (paired t-test, p=0.7036, n = 3) expression normalized to actin levels in iso31 animals under control and SD conditions.

Figure 3 with 1 supplement

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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 total sleep duration (B), sleep bout length (C) and bout number (D) for the nSyb-Gal4 control (black, n = 30), the UAS-HMS01617 control (blue, n = 32), and knockdown animals (nSyb >HMS01617, green, n = 32). (bar graphs show mean + standard deviation, one-way ANOVA with Tukey’s post-hoc test, *p<0.05, ***p<0.001, ****p<0.0001). (**E–H**) Baseline sleep upon knockdown with UAS-JF02142 (RNAi 2). (E) 24 hr sleep profile showing light/dark conditions on X-axis. Quantification of total sleep duration (F), sleep bout length (G) and bout number (H) for the nSyb-Gal4 control (black, n = 30), the UAS- JF02142 control (blue, n = 32), and knockdown animals (nSyb >JF02142, green, n = 32). The plotted nSyb-Gal4 control data is the same as in A-D, as the experiments were done simultaneously. (one-way ANOVA with Tukey’s post-hoc test, ***p<0.001, ****p<0.0001). (**I, J**) Recovery sleep upon knockdown with UAS-HMS01617 (RNAi 1). (I) 24 hr sleep profile of baseline and recovery days, and (J) the duration of sleep during ZT0-6 recovery period. nSyb-Gal4 control (black), the UAS-HMS01617 control (blue), and knockdown animals (nSyb >HMS01617, green). (n = 27 per genotype, one-way ANOVA with Tukey’s post-hoc test). (**K, L**) Recovery sleep upon knockdown with UAS-JF02142 (RNAi 2). (K) 24 hr sleep profile of baseline and recovery day, (L) duration of sleep during ZT0-6 recovery period (L). nSyb-Gal4 control (black), the UAS- JF02142 control (blue), and knockdown animals (nSyb >HMS01617, green). (n = 32 per genotype, one-way ANOVA with Tukey’s post-hoc test).

Figure 3—figure supplement 1

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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). (B) Activity Index for knockdown with UAS-JF02142 (RNAi 2) for the nSyb-Gal4 control (black; n = 30), the UAS-JF02142 control (blue; n = 32), and knockdown animals (nSyb >HMS01617, green; n = 32).

Figure 4 with 1 supplement

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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 (AANAT1-RNAi 2). Box and whisker plot as in Figure 2F. One-way ANOVA with Tukey’s post-hoc test, n = 7–9 per genotype. (**B, C**) Baseline sleep upon knockdown with UAS-HMS01617 (RNAi 1). 24 hr sleep profile (B), and total sleep duration (C) for the Alrm-Gal4 control (black; n = 32), the UAS-HMS01617 control (blue; n = 26), and knockdown animals (Alrm >HMS01617, green; n = 32), (one-way ANOVA with Tukey’s post-hoc test). (**D, E**) Baseline sleep upon knockdown with UAS-JF02142 (RNAi 2). 24 hr sleep profile (D), and total sleep duration (E) for the Alrm-Gal4 control (black; n = 32), the UAS-JF02142 control (blue; n = 32), and knockdown animals (Alrm >JF02142, green; n = 30). The plotted Alrm-Gal4 control data is the same as in B and C, as the experiments were done simultaneously, (one-way ANOVA with Tukey’s post-hoc test). (**F, G**) Recovery sleep upon knockdown with UAS-HMS01617 (RNAi 1). 24 hr sleep profile of baseline day and recovery day (F), and the duration of sleep during ZT0-6 recovery period (G) for the Alrm-Gal4 control (black), the UAS-HMS01617 control (blue), and knockdown animals (Alrm >HMS01617, green). (n = 31 per genotype, Kruskal–Wallis one-way ANOVA with Dunn’s post-hoc test, ****p<0.0001). (**H, I**) Recovery sleep upon knockdown with UAS-JF02142 (RNAi 2). 24 hr sleep profile of baseline day and recovery day (H), and the duration of sleep during ZT0-6 recovery period (I) for the Alrm-Gal4 control (black), the UAS-JF02142 control (blue), and knockdown animals (Alrm >HMS01617, green). (n = 32 per genotype, error bars are mean + standard deviation, one-way ANOVA with Tukey’s post-hoc test, ****p<0.0001). (J) HPLC-MS measurement of serotonin (Mann-Whitney t-test) and dopamine (Mann-Whitney t-test) in Alrm-Gal4 control (black; n = 7 for dopamine; n = 5 for serotonin) and Alrm >JF02142 (green; n = 6) fly brains under sleep deprivation (SD) conditions. For UAS controls (not shown), some samples fell below the limit of detection, leaving too few data points for robust statistical analysis. Box and whisker plots show 25–75% interquartile range (box), minimum and maximum (whiskers), median (horizontal line in box), and mean (+). (K) AANAT1 levels in astrocyte cell bodies normalized to nuRFP at ZT12,15,18,21 and 24 time-points. (n = 3 per time-point, 10 cells per sample, mean+ SEM).

Figure 4—figure supplement 1

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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). (B) Activity Index for knockdown with UAS-JF02142 (RNAi 2) for the Alrm-Gal4 control (black; n = 32), the UAS-JF02142 control (blue; n = 32), and knockdown animals (Alrm >JF02142, green; n = 30). (**C, D**) Nighttime sleep (C) and activity index (D) for *AANAT1^lo/+* heterozygotes (gray; n = 16) and *AANAT1^lo* homozygous mutants (red; n = 15). (Error bars are mean + standard deviation, Mann-Whitney t-test, p=0.0003). (E) Recovery sleep measured as the duration of sleep during ZT0-6 recovery period for *AANAT1^lo*/+ heterozygous (gray; n = 32) and *AANAT1^lo* homozygous mutants (red; n = 26). (Error bars are mean + standard deviation, Mann-Whitney t-test, p=0.0131). (F) Recovery sleep upon overexpression with UAS-AANAT1. The duration of sleep during ZT0-6 recovery period for the Alrm-Gal4 control (black), the UAS-AANAT1 control (blue), and overexpression animals (Alrm >AANAT1, brown). (n = 96 per genotype, error bars are mean + standard deviation, Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test, ****p<0.0001). (**G–I''**) Single slices of IHC for AANAT1 (magenta) and Alrm >nuRFP (green) showing AANAT1 expression above background levels (yellow arrowheads) in astrocytes during pupal development time 48 hr after puparium formation ( APF) (**G–G''**), 72 APF (**H–H''**) and 96 APF (**I–I''**). White arrowheads indicate astrocytes that do not express AANAT1. Scale bars in G'',H'',I''=20 μm. (**J–K**) Recovery sleep after adult-specific knockdown of AANAT1 with UAS-HMS01617 (RNAi 1) in astrocytes. 24 hr sleep profile of baseline and recovery day (J) and the duration of sleep during ZT0-6 recovery period (K). The duration of sleep during ZT0-6 recovery period (K) for the Eaat1-Gal4;tubGal80^ts control (black, n = 42), the UAS-HMS01617 control (blue, n = 46), and knockdown animals (Eaat1-Gal4;tubGal80^ts>HMS01617, green, n = 44). Kruskal–Wallis one-way ANOVA with Dunn’s post-hoc test, **p<0.01, ***p<0.001.

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^*; wg^Sp-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} VGlut^MI04979	Bloomington Drosophila Stock Center	RRID:BDSC_38078	FlyBase genotype: y¹w^*; Mi{MIC}VGlut^MI04979
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}Myo31DF^T2
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)	AANAT1^lo	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)	tubGal80^ts	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