1. Neuroscience

Drosophila TRPγ is required in neuroendocrine cells for post-ingestive food selection

  1. Subash Dhakal
  2. Qiuting Ren
  3. Jiangqu Liu
  4. Bradley Akitake
  5. Izel Tekin
  6. Craig Montell  Is a corresponding author
  7. Youngseok Lee  Is a corresponding author
  1. Department of Bio and Fermentation Convergence Technology, Kookmin University, Republic of Korea
  2. Department of Biological Chemistry, The Johns Hopkins University School of Medicine, United States
  3. Neuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, United States
https://doi.org/10.7554/eLife.56726
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Cite this article
  1. Subash Dhakal
  2. Qiuting Ren
  3. Jiangqu Liu
  4. Bradley Akitake
  5. Izel Tekin
  6. Craig Montell
  7. Youngseok Lee
(2022)
Drosophila TRPγ is required in neuroendocrine cells for post-ingestive food selection
eLife 11:e56726.
https://doi.org/10.7554/eLife.56726
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7 figures, 2 videos, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
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Characterization of TRPγ as a post-ingestion sensor using two-way feeding assays.

(A) Binary food choice assay showing preferences of control (w1118) and indicated flies (both males and females) to nutritive sugar (50 mM D-glucose) versus non-nutritive, but sweeter sugar (200 mM …

Figure 1—figure supplement 1
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Tip recordings, and behavioral assays indicating that trpγ functions in diuretic hormone 44 (Dh44) neurons, and not in peripheral neurons.

(A) Frequencies of action potentials produced by electrophysiological recordings performed on L6 sensilla from labella of the indicated flies in response to 50 mM D-glucose or 200 mM L-glucose, …

Figure 2 with 1 supplement
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GCaMP responses of diuretic hormone 44 (Dh44) neurons to D-glucose or L-glucose.

Changes in GCaMP6s fluorescence in Dh44 neurons exposed to D-glucose or L-glucose. Brains were isolated from control, trpγ1/+, and trpγ1 male flies carrying the Dh44-GAL4 and UAS-GCaMP6s, and …

Figure 2—figure supplement 1
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Quantification of GCaMP6s responses of diuretic hormone 44 (DH44) neurons to stimulation with 20 mM D-glucose.

Peak amplitudes (ΔF/F) were calculated by subtracting the pre-stimulation baseline from the sugar-evoked (20 mM D-glucose) peak value. The pre-stimulation baselines were calculated as the average of …

Figure 3 with 1 supplement
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Voltage imaging of diuretic hormone 44 (DH44) neurons with ASAP2s.

The Dh44-Gal4 and UAS-ASAP2s transgenes were introduced into the control and trpγ 1 backgrounds. The DH44 neurons were activated by D-glucose or L-glucose in ex vivo brain preparations. (A–C) …

Figure 3—figure supplement 1
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Quantification of intracellular anti-diuretic hormone 44 (DH44) signals in DH44 neurons after stimulation with different concentrations of D-glucose, L-glucose, and D-fructose.

(A) Quantification of immunofluorescence intensities (arbitrary units) produced by intracellular DH44 peptide probed with anti-DH44 with the indicated concentrations of D-glucose, L-glucose, and …

Figure 4 with 1 supplement
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Sugars and glycogen levels under sated and starvation conditions.

(A) Glucose levels in the hemolymph. n = 7–10. (B) Trehalose and glucose levels in whole-body extracts. n = 6–7. (C) Glycogen levels in whole-body extracts. n = 5–8. Means ± SEMs. Single-factor …

Figure 4—figure supplement 1
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Sugars and glycogen levels under fed and starved conditions, and survival of flies under starvation condition.

(A) Trehalose and glucose levels in whole-body extracts after RNAi knockdown of trpγ under control of the Dh44-GAL4 in sated flies (5 hr starvation) or in flies starved for 18 hr. n = 5. (B) …

Figure 5 with 1 supplement
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Requirement for trpγ in diuretic hormone 44 (DH44) neurons to control consumption.

(A–B) Confocal images of trpγ reporter expression (trpγG4/UAS-mCD8::GFP) in (A) the crop and (B) the intestine after staining the tissues with anti-GFP. Scale bars represent 25 µm. (C–G) …

Figure 5—figure supplement 1
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Internal food consumption.

Measurements of internalized food levels after feeding starved males 5% D-glucose or 5% L-glucose for 30 min. n = 8. Means ± SEMs. Unpaired Student’s t-tests were used for comparing two sets of …

Figure 6 with 1 supplement
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Crop extension and contraction rates.

(A) Representative images of crop sizes scored on a 1–5 scale after feeding for 12 hr 1% agarose containing 5% sucrose and blue food coloring. The images were acquired with an isolution IMTcam3 …

Figure 6—figure supplement 1
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Scoring crop sizes after feeding on different sugars.

Male flies were fed 5% of the indicated sugars for 12 hr before scoring the percentages of each crop size (1–5 scale). (A) Crop sizes after feeding on 5% sucrose. n = 5 (total 80 flies). (B) Crop …

Figure 7 with 1 supplement
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Rates and quantities of defecation.

(A) Cumulative defecation rates of male flies. n = 9. (B) Defecation rates of flies after 1 hr. n = 8–12. (C) Quantification of excreta size (area) of indicated male flies. n = 20. (D) …

Figure 7—figure supplement 1
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Body weight measurements.

Body weights of the indicated males under fed (no starvation) (blue data points) and 24 hr starvation (salmon colored data points) conditions were measured by selecting 10 (3- to 4-day-old) males of …

Videos

Video 1
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Control fly displaying a proboscis extension response (PER) to 200 mM L-glucose after 18 hr starvation.
Video 2
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trpγ1 mutant fly displaying a proboscis extension response (PER) to 200 mM L-glucose after 18 hr starvation.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)trpγ1Akitake et al., 2015 
Genetic reagent (Drosophila melanogaster)trpγG4Akitake et al., 2015 
Genetic reagent (Drosophila melanogaster)trpγ1,UAS- trpγ/CyOAkitake et al., 2015 
Genetic reagent (Drosophila melanogaster)trpγ1;g(trpγ)Akitake et al., 2015 
Genetic reagent (Drosophila melanogaster)Dh44MiBloomington Drosophila Stock Centerstock # 1454; RRID:BDSC_1454Provided by Y Kim (GIST) and Dr GS Suh (KAIST)
Genetic reagent (Drosophila melanogaster)Dh44-GAL4Bloomington Drosophila Stock Centerstock # 1453; RRID:BDSC_1453Provided by Y Kim (GIST) and Dr GS Suh (KAIST)
Genetic reagent (Drosophila melanogaster)UAS-hid/CyOBloomington Drosophila Stock Centerstock # 65403; RRID:BDSC_65403 
Genetic reagent (Drosophila melanogaster)UAS-mCD8::GFPBloomington Drosophila Stock Centerstock # 5130; RRID:BDSC_5130 
Genetic reagent (Drosophila melanogaster)UAS-nls::tdtomatoKnapp et al., 2015Provided by S Marella
Genetic reagent (Drosophila melanogaster)UAS-dicer2;UAS-trpγRNAiAkitake et al., 2015Stock # 107656VDRC-107656 
Genetic reagent (Drosophila melanogaster)iav-GAL4Kwon et al., 2010 
Genetic reagent (Drosophila melanogaster)dilp2-GAL4Rulifson et al., 2002Provided by EJ Rulifson
Genetic reagent (Drosophila melanogaster)Gr64abJiao et al., 2007 
Genetic reagent (Drosophila melanogaster)trpA11Kwon et al., 2008 
Genetic reagent (Drosophila melanogaster)amo1Watnick et al., 2003 
Genetic reagent (Drosophila melanogaster)trpml2Venkatachalam et al., 2008 
Genetic reagent (Drosophila melanogaster)trp343Wang et al., 2005Originally isolated from W Pak lab
Genetic reagent (Drosophila melanogaster)pyx3Lee et al., 2005 
Genetic reagent (Drosophila melanogaster)wtrwexWang et al., 2005 
Genetic reagent (Drosophila melanogaster)trpl29134Niemeyer et al., 1996 
Genetic reagent (Drosophila melanogaster)pain2Tracey et al., 2003 
Genetic reagent (Drosophila melanogaster)nan36aKim et al., 2003 
Genetic reagent (Drosophila melanogaster)UAS-GCaMP6sBloomington Drosophila Stock Centerstock # 77131; RRID:BDSC_77131 
Genetic reagent (Drosophila melanogaster)UAS-dsRedBloomington Drosophila Stock Centerstock # 59853; RRID:BDSC_59853 
Genetic reagent (Drosophila melanogaster)20xUAS-ASAP2sBloomington Drosophila Stock Centerstock# 76247;
RRID:BDSC_76247		 
Genetic reagent (Drosophila melanogaster)UAS-Kir2.1Bloomington Drosophila Stock Centerstock # 6596; RRID:BDSC_6596 
Genetic reagent (Drosophila melanogaster)iav3621Bloomington Drosophila Stock Centerstock # 24768; RRID:BDSC_24768Generated by Dr P Salvaterra
Genetic reagent (Drosophila melanogaster)tubulin-GAL80tsBloomington Drosophila Stock Centerstock # 7017; RRID:BDSC_7017 
AntibodyMouse anti-GFP (mouse monoclonal)Molecular ProbesA-11120(1:1000, 2 μL)
AntibodyRabbit anti-DsRed (rabbit polyclonal)Clontech632496(1:1000, 2 μL)
AntibodyGoat anti-mouse IgG (mouse polyclonal)Thermo Fisher/InvitrogenA-11031Alexa Fluor 488 (1:200, 10 μL)
AntibodyGoat anti-rabbit IgG (rabbit polyclonal)Thermo Fisher/InvitrogenA-11034Alexa Fluor 568 (1:200, 20 μL)
AntibodyRabbit anti-DH44 (rabbit polyclonal)Cabrero et al., 2002(1:500, 15 μL)
Commercial assay or kitGlucose (HK) Assay KitSigma-AldrichSigma: G3293 
Chemical compound, drugBrilliant blue FCFTakeda-Wako Pure Chemical Industry LtdCat. 3844-45-9 
Chemical compound, drugParaformaldehydeElectron Microscopy SciencesCat. 15710 
Chemical compound, drugSulforhodamine BSigma-AldrichCat. 3520-42-1 
Chemical compound, drugSucroseSigma-AldrichCat. 57-50-1 
Chemical compound, drugD-(+)-GlucoseSigma-AldrichCat. 50-99-7 
Chemical compound, drugD-(-)-FructoseSigma-AldrichCat. 57-48-7 
Chemical compound, drugD-(+)-MaltoseSigma-AldrichCat. 6363-53-7 
Chemical compound, drugD-(+)-TrehaloseSigma-AldrichCat. 6138-23-4 
Chemical compound, drugKClSigma-AldrichCat. 7447-40-7 
Chemical compound, drugHydrochloric acidSamchunCat. 7647-01-0 
Chemical compound, drugEthanolMerckCat. 64-17-5 
Chemical compound, drugTrehalase from porcine kidneySigma-AldrichCat. 9025-52-9 
Chemical compound, drugAmyloglucosidase from AspergillusSigma-AldrichCat. 9032-08-0 

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/56726/elife-56726-transrepform1-v2.pdf
Source data 1

Detailed statistics for all data.

https://cdn.elifesciences.org/articles/56726/elife-56726-data1-v2.docx

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