Modulation of flight and feeding behaviours requires presynaptic IP3Rs in dopaminergic neurons
- National Centre for Biological Sciences, TIFR, India
Figures
Figure 1
Generation and characterisation of a dominant negative IP3R (IP3RDN).
(A) Domain organisation of the Drosophila IP3R. The Arginine (R) and Lysine (K) residues mutated to Glutamine (Q) to generate a dominant-negative IP3R (IP3RDN) are shown (top). Alignment of the Droso…
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Figure 1—source data 1
Western Blots for IP3R upon pan-neuronal expression of IP3RDN and IP3R WT in adult heads.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig1-data1-v1.pptx
Figure 2 with 2 supplements
The IP3R is required in central dopaminergic neurons for maintaining long flight bouts.
(A) Flight deficits observed in flies expressing IP3R RNAi (dsitpr), IP3RDN (ItprDN) and IP3RWT (Itpr+) across all dopaminergic and TH-expressing cells (TH) as well as a subset of dopaminergic …
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Figure 2—source data 1
Flight duration for flies upon perturbing IP3R signaling in dopaminergic neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig2-data1-v1.xlsx
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Figure 2—source data 2
Flight duration of itpr mutant flies after overexpressing a wild type itpr transgene in various dopaminergic subs.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig2-data2-v1.xlsx
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Figure 2—source data 3
Flight duration of flies upon perturbing IP3R signaling in PPL1 neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig2-data3-v1.xlsx
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Figure 2—source data 4
Flight duration of flies upon perturbing IP3R signaling pan neuronally.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig2-data4-v1.xlsx
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Figure 2—source data 5
Flight duration of itpr mutant flies after overexpressing a wild type itpr transgene in various neuronal subsets in a short flight assay.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig2-data5-v1.xlsx
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Figure 2—source data 6
Flight duration of flies upon perturbing IP3R signaling in subsets of PPL1 .neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig2-data6-v1.xlsx
Figure 2—figure supplement 1
Perturbation of IP3R signaling in neurons affects the duration of flight bouts.
(A) Box plots represent flight deficits observed upon perturbing IP3R signaling after expressing IP3R RNAi (dsitpr), IP3RDN (ItprDN) and IP3RWT (Itpr+) across all classes of neurons with nsybGAL4. (B…
Figure 2—video 1
Flight defect in THD’> ItprDN (Right) as compared to control (THD’/+, Left).
Figure 3 with 1 supplement
Adult flight phenotypes arise from late pupal expression of the IP3R and mAChR.
(A) Box plots of flight bout durations in adults after temporal expression of IP3RDN in THD’ neurons (THD’; TubGAL80ts > ItprDN) by transferring them to 29°C at the indicated stages of development. …
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Figure 3—source data 1
Flight duration of flies upon overexpression of IP3RDN at different stages of development.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig3-data1-v1.xlsx
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Figure 3—source data 2
Flight duration of itpr mutant flies after overexpressing a wild type itpr transgene at different stages of development.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig3-data2-v1.xlsx
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Figure 3—source data 3
Western blots of IP3R.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig3-data3-v1.pptx
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Figure 3—source data 4
Flight duration of flies upon knockdown of mAChR in dopaminergic neurons at different stages of development.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig3-data4-v1.xlsx
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Figure 3—source data 5
Flight duration of flies upon knockdown of mAChR in PPL1-γ2α′1 neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig3-data5-v1.xlsx
Figure 3—figure supplement 1
Expression of IP3RDN and mAChR RNAi in THD’ and MB296B marked neurons.
(A) Expression of IP3RDN does not change the number of PPL1 neurons. Cells were quantified from both hemi-segments of N=6 brains. (B) PPL1- γ2α′1 neurons marked by MB296BGAL4 are shown (white …
Figure 4 with 3 supplements
Synaptic vesicle recycling and IP3R function are required in PPL1 dopaminergic neurons for adult flight and feeding.
(A) Quantification of flight deficits observed upon blocking synaptic vesicle recycling by expression of a temperature-sensitive Dynamin mutant, Shibirets in THD’ and MB296B cells. n≥30, ***p< …
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Figure 4—source data 1
Flight duration of flies upon expression of Shibirets in dopaminergic neurons at different developmental stages.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig4-data1-v1.xlsx
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Figure 4—source data 2
Food seeking index of starved male flies after overexpression of IP3RDN in dopaminergic neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig4-data2-v1.xlsx
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Figure 4—source data 3
Food seeking index of starved male flies upon knockdown of mAChR in dopaminergic neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig4-data3-v1.xlsx
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Figure 4—source data 4
Flight duration of flies upon expression of Shibirets in dopaminergic neurons, grown at 25°C.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig4-data4-v1.xlsx
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Figure 4—source data 5
Food seeking index of fed male flies after overexpression of IP3RDN in dopaminergic neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig4-data5-v1.xlsx
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Figure 4—source data 6
Food seeking index of fed male flies upon knockdown of mAChR in dopaminergic neurons.
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig4-data6-v1.xlsx
Figure 4—figure supplement 1
Flight is unaffected and food seeking behaviour is normal in control genotypes and conditions.
(A) Quantification of flight bouts upon expressing temperature-sensitive dynamin mutant Shibirets in THD’ and MB296B cells when flies were grown at the permissive temperature of 25°C. n≥30, n.s., …
Figure 4—video 1
Food-seeking behaviour in a control fly (MB296BGAL4/+).
Figure 4—video 2
Food-seeking behaviour upon expression of IP3RDN in MB296B cells.
Figure 5 with 1 supplement
Carbachol-stimulated Dopamine release at the axonal terminals of PPL1-γ2α′1 neurons is attenuated by expression of IP3RDN.
(A) Representative images of a presynaptic marker synaptotagmin GFP (syt.eGFP) in lobes of the Mushroom body (top). Quantification of syt.eGFP fluorescence normalized to mRFP is shown below in the …
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Figure 5—source data 1
GRABDA fluorescence (ΔF/F) traces from axonal termini of MB296BGAL4 marked DANs in individual brains (Black -MB296B GAL4>UAS GRABDA; Red-MB296B GAL4>UAS ItprDN; UAS GRABDA).
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig5-data1-v1.pptx
Figure 5—figure supplement 1
Carbachol evoked Ca2+ responses in the soma of PPL1- γ2α′1 DANs marked by MB296BGAL4.
(A) Carbachol stimulated change in GCaMP6m fluorescence (ΔF/F) in PPL1- γ2α′1 cells. Representative images of GCaMP6m fluorescence at the indicated time points (top panel). Warmer colors denote …
Figure 6 with 1 supplement
Optimal excitability of PPL1- γ2α′1 dopaminergic neurons requires the IP3R.
(A) Optogenetic activation of PPL1- γ2α′1 DANs, with the red light activated Channelrhodopsin variant Chrimson, is attenuated by expression of IP3RDN. Representative images of MB296BGAL4 marked DANs …
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Figure 6—source data 1
GCaMP6m fluorescence (ΔF/F) traces inMB296BGAL4 marked DANs after activation by Chrimson in individual brains (Black -MB296B GAL4>UAS RFP; UAS GCaMP; Red-MB296B GAL4>UAS ItprDN; UAS GCaMP).
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig6-data1-v1.pptx
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Figure 6—source data 2
Arclight fluorescence (ΔF/F) traces in MB296BGAL4 marked DANs after KCl induced depolarisation in individual brains (Black -MB296B GAL4>UAS Arclight; Red-MB296B GAL4>UAS ItprDN; UAS Arclight).
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig6-data2-v1.pptx
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Figure 6—source data 3
GCaMP fluorescence (ΔF/F) traces inMB296BGAL4marked DANs after KCl induced depolarisation in individual brains (Black -MB296B GAL4>UAS GCaMP; Red-MB296B GAL4>UASItprDN; UAS GCaMP).
- https://cdn.elifesciences.org/articles/62297/elife-62297-fig6-data3-v1.pptx
Figure 6—figure supplement 1
Perturbation of IP3R signaling in PPL1- γ2α′1 cells reduced KCl-evoked calcium response.
(A) KCl-evoked activation of PPL1- γ2α′1 DANs is attenuated by expression of IP3RDN. Representative images of MB296BGAL4 marked DANs of the indicated genotypes are shown with changes in GCaMP6m …
Figure 7
Schematic showing neuronal properties regulated by IP3/Ca2+ signals in central dopaminergic neurons for flight and food search behaviour in Drosophila melanogaster.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | nSybGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_51635 | |
| Genetic reagent (D. melanogaster) | UAS GCaMP6m | Bloomington Drosophila Stock Center | RRID:BDSC_42750 | |
| Genetic reagent (D. melanogaster) | UAS GCaMP6m | Bloomington Drosophila Stock Center | RRID:BDSC_42748 | |
| Genetic reagent (D. melanogaster) | UAS Arclight | Bloomington Drosophila Stock Center | RRID:BDSC_51056 | |
| Genetic reagent (D. melanogaster) | UAS mCD8GFP | Bloomington Drosophila Stock Center | RRID:BDSC_5130 | |
| Genetic reagent (D. melanogaster) | UAS Dicer | Bloomington Drosophila Stock Center | RRID:BDSC_24648 | |
| Genetic reagent (D. melanogaster) | UAS Chrimson | Bloomington Drosophila Stock Center | RRID:BDSC_55137 | |
| Genetic reagent (D. melanogaster) | UAS syt.eGFP | Bloomington Drosophila Stock Center | RRID:BDSC_6926 | |
| Genetic reagent(D. melanogaster) | UAS RFP | Bloomington Drosophila Stock Center | RRID:BDSC_32218 | |
| Genetic reagent (D. melanogaster) | MB058BGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_68278 | |
| Genetic reagent (D. melanogaster) | MB296BGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_63308 | |
| Genetic reagent (D. melanogaster) | MB304BGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_68367 | |
| Genetic reagent (D. melanogaster) | MB320CGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_68253 | |
| Genetic reagent (D. melanogaster) | MB630BGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_68334 | |
| Genetic reagent (D. melanogaster) | MB438BGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_68326 | |
| Genetic reagent (D. melanogaster) | MB504BGAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_68329 | |
| Genetic reagent (D. melanogaster) | UAS mAChR RNAi (dsmAChR) | Vienna Drosophila Resource Center RRID:SCR_013805 | VDRC_101407 | |
| Genetic reagent (D. melanogaster) | UAS itprRNAi (dsitpr) | National Institute of Genetics | NIG_1063 R-2 | |
| Genetic reagent (D. melanogaster) | UAS Itpr+ | Venkatesh et al., 2001 | RRID:BBSC_30742 | |
| Genetic reagent (D. melanogaster) | THGAL4 | Friggi-Grelin et al., 2003 (DOI:10.1002/neu.10185) | Gift from Serge Birman (CNRS, ESPCI Paris Tech, France) | |
| Genetic reagent (D. melanogaster) | THD’ GAL4 | Liu et al., 2012 (DOI:10.1016/j.cub.2012.09.008) | Gift from Mark N Wu (Johns Hopkins University, Baltimore) | |
| Genetic reagent (D. melanogaster) | THD1 GAL4 | Liu et al., 2012 (DOI:10.1016/j.cub.2012.09.008) | Gift from Mark N Wu (Johns Hopkins University, Baltimore) | |
| Genetic reagent (D. melanogaster) | THC’ GAL4 | Liu et al., 2012 (DOI:10.1016/j.cub.2012.09.008) | Gift from Mark N Wu (Johns Hopkins University, Baltimore) | |
| Genetic reagent (D. melanogaster) | UAS GRABDA | Sun et al., 2018 (DOI:10.1016/j.cell.2018.06.042) | Gift from Yulong Li (Peking University School of Life Sciences, Beijing, China) | |
| Genetic reagent (D. melanogaster) | UAS Shibirets | Kitamoto, 2001 (DOI:10.1002/neu.1018) | Gift from Toshihiro Kitamoto (University of Iowa, Carver College of Medicine, Iowa) | |
| Genetic reagent (D. melanogaster) | vGlutVGN6341GAL4 | Syed et al., 2016 (DOI:10.7554/eLife.11572) | Gift from K. Vijayraghavan (NCBS, India) | |
| Genetic reagent (D. melanogaster) | UAS mitoGCaMP | Lutas et al., 2012 (DOI:10.1534/g3.111.001586) | Gift from Fumiko Kawasaki (Pennsylvania State University, Pennsylvania) | |
| Genetic reagent (D. melanogaster) | UAS TubGAL80ts | Pathak et al., 2015 (DOI:10.1523/JNEUROSCI.1680–15.2015) | Generated by Albert Chiang, NCBS, Bangalore, India | |
| Genetic reagent (D. melanogaster) | UAS ItprDN | This paper | Transgenic Drosophila with a Dominant negative Drosophila IP3R cDNA | |
| Genetic reagent (D. melanogaster) | itpr gene mutant (itprug3) | Joshi et al., 2004 (DOI:10.1534/genetics.166.1.225) | RRID:BDSC_30738 | |
| Genetic reagent (D. melanogaster) | itpr gene mutant (itprka1091) | Joshi et al., 2004 (DOI:10.1534/genetics.166.1.225) | RRID:BDSC_30739 | |
| Antibody | Rabbit anti GFP (polyclonal) | Life Technologies, Thermo Fisher | RRID:AB_221570,Cat # A-6455 | IHC: 1:10,000 |
| Antibody | Mouse anti-bruchpilot (monoclonal) | Wagh et al., 2006 (DOI:10.1016/j.neuron.2006.02.008) | Gift from Eric Buchner, University of Wuerzburg, Germany (dilution 1:150) | |
| Antibody | Anti-mouse Alexa Fluor 568 (polyclonal) | Life Technologies, ThermoFisher Scientific | Cat# A-11004, RRID:AB_2534072 | IHC: 1:400 |
| Antibody | Anti-rabbit Alexa Fluor 488 (polyclonal) | Life Technologies, ThermoFisher Scientific | Cat# A-11008, RRID:AB_143165 | IHC: 1:400 |
| Antibody | Rabbit anti-IP3R (polyclonal) | Agrawal et al., 2009 (DOI:10.1371/journal.pone.0006652) | IB-9075 | WB: 1:300; Gift from Ilya Bezprozvanny (UT South Western, USA). |
| Antibody | Mouse anti-spectrin (monoclonal) | Developmental Studies Hybridoma Bank | Cat# 3A9 (323 or M10-2), RRID:AB_528473 | WB: 1:50 |
| Antibody | Anti-mouse HRP (polyclonal) | Cell Signaling Technology | Cat# 7076, RRID:AB_330924 | WB: 1:3000 |
| Antibody | Anti-rabbit HRP (polyclonal) | ThermoFisher Scientific | Cat#32260, RRID:AB_1965959 | WB: 1:5000 |
| Chemical compound, drug | Low melt Agar | Invitrogen | Cat# 16520–050 | |
| Chemical compound, drug | Carbachol | Sigma Aldrich | Cat# C4382 | |
| Chemical compound, drug | All trans Retinal | Sigma Aldrich | Cat# R2500 | |
| Chemical compound, drug | EcoR1 | New England Biolabs | Cat# R0101S | Restriction enzyme |
| Chemical compound, drug | Xho1 | New England Biolabs | Cat# R0146S | Restriction enzyme |
| Chemical compound, drug | AatII | New England Biolabs | Cat# R0117S | Restriction enzyme |
| Chemical compound, drug | Eag1 | New England Biolabs | Cat# R0505S | Restriction enzyme |
| Strain, strain background (Escherichia coli) | Sure Competent cells | Stratagene, Agilent Technologies | Cat# 200238 | Maintained in the lab |
| Commercial assay or kit | Quick Ligation Kit | New England Biolabs | Cat# M2200S | |
| Commercial assay or kit | WesternBright ECL kit | Advansta | Cat# K-12045-D20 | |
| Commercial assay or kit | QuikChange II XL Site-Directed Mutagenesis Kit | Agilent | Cat# 200522 | |
| Software, algorithm | Origin 8 | Origin lab | RRID:SCR_014212 | |
| Software, algorithm | Fiji/ImageJ | National Institutes of Health | RRID:SCR_002285 |
