Sleep-promoting effects of threonine link amino acid metabolism in Drosophila neuron to GABAergic control of sleep drive
- Ulsan National Institute of Science and Technology, Republic of Korea
Figures
Figure 1 with 8 supplements
Dietary threonine promotes sleep and facilitates sleep onset.
(A) Wild-type male flies were individually loaded on to 5% sucrose food containing 17.5 mM of each amino acid (day 0) and entrained in LD cycles at 25°C. Total sleep amount (top) and latency to …
Figure 1—figure supplement 1
Dietary threonine increases the number of sleep bouts but decreases waking activities.
Sleep behaviors in wild-type male flies were analyzed similarly to the data presented in Figure 1A. Waking activity (top), the number of sleep bouts (middle), and averaged sleep bout length (ABL, …
Figure 1—figure supplement 2
Sleep-promoting effects of dietary threonine (SPET) are dose-dependent and observed in both male and female flies.
(A and B) Sleep behaviors in individual male (A) or female (B) flies were analyzed similarly to the data presented in Figure 1A. Error bars indicate mean ±95% CI (n = 11–213). *p<0.05, ***p<0.001 to …
Figure 1—figure supplement 3
Transgenic silencing of sensory neurons that express either gustatory or olfactory receptors does not abolish SPET.
Sleep behaviors in individual male flies were analyzed similarly to the data presented in Figure 1A. Error bars indicate mean ±95% CI (n = 14–38). Two-way ANOVA detected significant effects of …
Figure 1—figure supplement 4
Wild-type flies fed protein-rich food display SPET comparably to those fed sucrose-based food.
Wild-type flies were loaded on to nutrient-rich cornmeal-yeast-agar food containing additional protein sources (e.g., cornmeal, yeast) along with the indicated concentration of threonine supplement …
Figure 1—figure supplement 5
A video-based sleep analysis validates SPET in threonine-fed flies.
(A) The DAM system counts when a fly in the glass tube crosses an infrared (IR) beam sensor. The total number of beam crosses per minute is continuously recorded as a proxy for the locomotor …
Figure 1—figure supplement 6
Dietary threonine does not impair general locomotion.
Wild-type male flies were loaded on to 5% sucrose food containing each amino acid (day 0) and entrained in LD cycles at 25°C. Individual flies were transferred into each arena (diameter x height = 35…
Figure 1—figure supplement 7
Dietary threonine induces a higher sleep drive.
Wild-type male flies were loaded on to 5% sucrose food containing the indicated amount of threonine (day 0) and entrained in LD cycles at 25°C. Control- and threonine-fed flies were exposed to a 1 …
Figure 1—figure supplement 8
Genetic loss of Lk or Lkr function does not affect SPET.
Sleep behaviors in individual male flies trans-heterozygous or heterozygous for Lk or Lkr mutant alleles were analyzed similarly to the data presented in Figure 1A. Two-way ANOVA detected comparable …
Figure 2 with 2 supplements
Circadian rhythms and clock-dependent control of sleep onset are dispensable for SPET.
(A) Arrhythmic clock mutants were loaded on to 5% sucrose food containing the indicated amount of threonine (day 0) and entrained in LD cycles at 25°C. Sleep behaviors in individual female flies …
Figure 2—figure supplement 1
Genetic loss of Rdl function does not suppress SPET.
Female mutants trans-heterozygous or heterozygous for hypomorphic Rdl alleles (RdlMDRR or Rdl1) were loaded on to 5% sucrose food containing the indicated amount of threonine (day 0) and entrained …
Figure 2—figure supplement 2
Constant light (LL) strongly dampens daily rhythms in sleep-wake cycles and sleep latency, but SPET is detectable in LL.
Sleep behaviors in individual flies were analyzed similarly to the data presented in Figure 2C. Sleep latency was measured at each time-point during LD or LL cycle on day 4. Two-way ANOVA detected …
Figure 3 with 1 supplement
Genetic or pharmacological elevation of GABA suppresses SPET.
(A) GABA-T trans-heterozygous mutants were resistant to SPET. Sleep behaviors in individual male flies were analyzed similarly to the data presented in Figure 1A. Two-way ANOVA detected significant …
Figure 3—figure supplement 1
Transgenic overexpression of wild-type GABA-T partially rescues baseline sleep and SPET in GABA-T mutants.
(A) Loss-of-function mutant alleles in the GABA-T locus. Upper or lower arrows indicate the direction of the insertional allelic transgenes in relative to the transcriptional start site in the GABA-T…
Figure 4 with 6 supplements
Down-regulation of metabotropic GABA transmission likely mediates SPET.
(A) Dietary threonine decreased the relative levels of select amino acids including GABA and glutamate. Wild-type male flies were loaded on to standard cornmeal-yeast-agar food containing either 0 …
Figure 4—figure supplement 1
Dietary threonine selectively elevates pyruvate levels but dietary pyruvate itself does not promote sleep.
(A) Quantitative analyses of energy metabolites in fly heads. Wild-type male flies were loaded on to standard cornmeal-yeast-agar food containing either 0 mM (control) or 50 mM threonine, and then …
Figure 4—figure supplement 2
A metabolic pathway of serine, glycine, and threonine.
A schematic diagram of enzymes (Drosophila homologs) and biochemical reactions in the metabolic pathway of serine, glycine, and threonine was adopted and modified from KEGG pathway database (http://w…
Figure 4—figure supplement 3
Glutamate supplement does not suppress SPET.
Wild-type flies were individually loaded on to 5% sucrose food containing the increasing amount of threonine (day 0) and entrained in LD cycles at 25°C. Where indicated, 25 mM glutamate was added to …
Figure 4—figure supplement 4
Dietary threonine elevates intracellular Ca2+ levels in a subset of GABAergic neurons.
(A) Confocal imaging of nuclear GFP (top, green) or a transcriptional reporter for intracellular Ca2+ (i.e., CaLexA-induced GFP) (bottom, green) expressed in GAD1-expressing GABAergic neurons by the …
Figure 4—figure supplement 5
Pan-neuronal depletion of metabotropic GABA receptor R1, but not R2, affects SPET.
Sleep behaviors in individual male flies were monitored similarly to the data presented in Figure 4C. Two-way ANOVA detected significant masking of SPET by the pan-neuronal RNAi on sleep amount …
Figure 4—figure supplement 6
Structural and functional relevance of alpha-ketobutyric acid, a threonine derivative, to GABA and GABA derivatives.
Space filling models were adopted from Wikipedia (https://en.wikipedia.org/).
Figure 5 with 2 supplements
Metabotropic GABA transmission in ellipsoid body R2 neurons contributes to SPET.
(A) A representative live-brain image of Epac1-camps (a transgenic FRET sensor for cAMP) expressed in R2 EB neurons by 58H05-Gal4 driver (left). An inverse correlation between intracellular cAMP …
Figure 5—figure supplement 1
R2 EB neurons are GABA-ceptive.
(A) A transgenic FRET sensor for cAMP (Epac1-camps) was expressed in R2 EB neurons by 58H05-Gal4 driver. Whole brains were dissected out from transgenic female flies and transferred to an imaging …
Figure 5—figure supplement 2
Transgenic depletion of metabotropic GABA receptor R1 in R2 EB neurons does not affect SPET.
Sleep behaviors in individual male flies were monitored similarly to the data presented in Figure 4C. Two-way ANOVA detected no significant masking of SPET on sleep amount or sleep latency by the …
Figure 6 with 1 supplement
Dietary threonine rescues short-term memory in dumb mutants with memory deficit in a sleep-dependent manner.
(A) An experimental design of the short-term memory (STM) test after three cycles of training on aversive phototaxis suppression. Wild-type (Canton S) or dumb2 mutant flies were individually loaded …
Figure 6—figure supplement 1
Dietary threonine rescues short-term memory in rutabaga mutants with memory deficit.
Wild-type or rutabaga mutant flies (rut2080) were fed control or threonine-containing food for 3 days in LD cycles at 25°C. Aversive phototaxic suppression was then tested in individual male flies …
Figure 7
Genetic suppression of threonine 3-dehydrogenase elevates endogenous threonine levels and facilitates sleep onset.
(A) A threonine metabolism catalyzed by threonine 3-dehydrogenase (CG5955). (B) A hypomorphic mutant allele of the P element insertion ([GS20382]) in the CG5955 locus. An amplicon used in …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | w1118 | Bloomington Drosophila Stock Center | RRID:BDSC_5905 | |
| Genetic reagent (D. melanogaster) | Canton S | Korea Drosophila Resource Center | Stock #K211 | |
| Genetic reagent (D. melanogaster) | CG5955GS20382 | Kyoto Drosophila Genomics and Genetics Resources | RRID:DGGR_201409 | |
| Genetic reagent (D. melanogaster) | Df(3L)BSC797 | Bloomington Drosophila Stock Center | RRID:BDSC_27369 | CG5955 deficiency |
| Genetic reagent (D. melanogaster) | Df(3L)BSC839 | Bloomington Drosophila Stock Center | RRID:BDSC_27917 | CG5955 deficiency |
| Genetic reagent (D. melanogaster) | rut2080 | Bloomington Drosophila Stock Center | RRID:BDSC_9405 | |
| Genetic reagent (D. melanogaster) | DA1dumb2 | Harvard Medical School | RRID: FlyBase_FBst1017920 | Dop1R1f02676 |
| Genetic reagent (D. melanogaster) | ELAV-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_458 | |
| Genetic reagent (D. melanogaster) | GAD1-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_51630 | |
| Genetic reagent (D. melanogaster) | 58H05-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_39198 | |
| Genetic reagent (D. melanogaster) | Gr5a-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_57591 | |
| Genetic reagent (D. melanogaster) | Gr33a-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_31425 | |
| Genetic reagent (D. melanogaster) | Gr66a-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_28801 | |
| Genetic reagent (D. melanogaster) | Orco-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_26818 | |
| Genetic reagent (D. melanogaster) | Lkc275 | Bloomington Drosophila Stock Center | RRID:BDSC_16324 | |
| Genetic reagent (D. melanogaster) | Df(3L)Exel6123 | Bloomington Drosophila Stock Center | RRID:BDSC_7602 | Lk deficiency |
| Genetic reagent (D. melanogaster) | Lkrc003 | Bloomington Drosophila Stock Center | RRID:BDSC_16250 | |
| Genetic reagent (D. melanogaster) | Df(3L)BSC557 | Bloomington Drosophila Stock Center | RRID:BDSC_25119 | Lkr deficiency |
| Genetic reagent (D. melanogaster) | per01 | PMID: 9630223 | RRID:BDSC_80917 | |
| Genetic reagent (D. melanogaster) | ClkJrk | PMID: 9630223 | RRID:BDSC_24515 | |
| Genetic reagent (D. melanogaster) | PDF-Gal4 | PMID: 10619432 | ||
| Genetic reagent (D. melanogaster) | UAS-ClkDN | Tanoue et al., 2004 | RRID:BDSC_36318 | |
| Genetic reagent (D. melanogaster) | RdlMDRR | Kyoto Drosophila Genomics and Genetics Resources | RRID:DGGR_106444 | |
| Genetic reagent (D. melanogaster) | Rdl1 | Kyoto Drosophila Genomics and Genetics Resources | RRID:DGGR_106453 | |
| Genetic reagent (D. melanogaster) | GABA-TPL | Bloomington Drosophila Stock Center | RRID:BDSC_19461 | GABATPL00338, null mutants |
| Genetic reagent (D. melanogaster) | GABA-TF | Harvard Medical School | RRID:FlyBase_FBst101711 | GABATf01602, hypomorphic |
| Genetic reagent (D. melanogaster) | GABA-TLL | Kyoto Drosophila Genomics and Genetics Resources | RRID:DGGR_141269 | GABATLL04492, hypomorphic |
| Genetic reagent (D. melanogaster) | UAS-GABA-T | Chen et al., 2015 | RRID:FlyBase_FBst0491743 | |
| Genetic reagent (D. melanogaster) | Df(3L)BSC731 | Bloomington Drosophila Stock Center | RRID:BDSC_26829 | GABA-T deficiency |
| Genetic reagent (D. melanogaster) | UAS-shibirets | Kitamoto, 2001 | ||
| Genetic reagent (D. melanogaster) | 30Y-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_30818 | |
| Genetic reagent (D. melanogaster) | TH-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_8848 | |
| Genetic reagent (D. melanogaster) | UAS-mLexA-VP16-NFAT | Masuyama et al., 2012 | RRID:BDSC_66542 | |
| Genetic reagent (D. melanogaster) | UAS-Epac1-camps | Bloomington Drosophila Stock Center | RRID:BDSC_25407 | |
| Genetic reagent (D. melanogaster) | UAS-CG5955RNAi#1 | Vienna Drosophila Resource Center | RRID:FlyBase_FBst0452036 | V15838 |
| Genetic reagent (D. melanogaster) | UAS-CG5955RNAi#2 | Bloomington Drosophila Stock Center | RRID:BDSC_64566 | |
| Genetic reagent (D. melanogaster) | UAS-Kir | PMID: 11222642 | ||
| Genetic reagent (D. melanogaster) | UAS-GABAB-R1RNAi#1 | Vienna Drosophila Resource Center | RRID:FlyBase_FBst0473313 | V101440 |
| Genetic reagent (D. melanogaster) | UAS-GABAB-R1RNAi#2 | Vienna Drosophila Resource Center | RRID:FlyBase_FBst0490977 | V330042 |
| Genetic reagent (D. melanogaster) | UAS-GABAB-R1RNAi#3 | Bloomington Drosophila Stock Center | RRID:BDSC_51817 | T51817 |
| Genetic reagent (D. melanogaster) | UAS-GABAB-R2RNAi#1 | Vienna Drosophila Resource Center | RRID:FlyBase_FBst0452890 | V1784 |
| Genetic reagent (D. melanogaster) | UAS-GABAB-R2RNAi#2 | Vienna Drosophila Resource Center | RRID:FlyBase_FBst0452896 | V1785 |
| Genetic reagent (D. melanogaster) | UAS-GABAB-R3RNAi#1 | Vienna Drosophila Resource Center | RRID:FlyBase_FBst0468888 | V50176 |
| Genetic reagent (D. melanogaster) | UAS-GABAB-R3RNAi#2 | Vienna Drosophila Resource Center | RRID:FlyBase_FBst0477558 | V108036 |
| Chemical compound, drug | EOS | Tokyo Chemical Industry | Cat. No. S0445 | |
| Chemical compound, drug | NipA | Sigma | Cat. No. 211672 | |
| Chemical compound, drug | THIP | Tocris | Cat. No. 0807 | Also known as gaboxadol, 2000x stock |
| Chemical compound, drug | SKF-97541 | Tocris | Cat. No. 0379 | 10000x stock |
| Chemical compound, drug | GABA | Acros | Cat. No. AC103280250 | 10x stock |
| Chemical compound, drug | Pyruvate | Sigma | Cat. No. P2256 | |
| Chemical compound, drug | Tetrodotoxin (TTX) | Alomone Labs | Cat. No. T-550 | 1000x stock |
| Chemical compound, drug | caffeine | Alfa Aesar | Cat. No. A10431 | 1000x stock |
| Antibody | Mouse anti-GFP, monoclonal | UC Davis/NIH NeuroMab Facility | RRID:AB_10671955 | 1:1000 dilution |
| Antibody | Rabbit anti-GABA, polyclonal | Sigma | RRID:AB_477652 | 1:2000 dilution |
| Antibody | Rabbit anti-TH, polyclonal | Millipore | RRID:AB_390204 | 1:1000 dilution |
| Antibody | Donkey anti-Mouse AF488 | Jackson Immunoresearch | RRID:AB_2340846 | 1:600 dilution |
| Antibody | Donkey anti-Rabbit AF594 | Jackson Immunoresearch | RRID:AB_2340621 | 1:600 dilution |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.40593.029
