A single pair of pharyngeal neurons functions as a commander to reject high salt in Drosophila melanogaster
- Department of Bio and Fermentation Convergence Technology, Kookmin University, Republic of Korea
- Neuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, United States
Figures
Figure 1 with 2 supplements
Testing requirements for Irs for avoiding high salt-containing food, and chemogenetic and optogenetic control of Ir60b gustatory receptor neurons (GRNs).
(A) Binary food choice assays (1 mM sucrose versus 5 mM sucrose and 300 mM NaCl) comparing 30 Ir mutants to the control strain (w1118) for high salt avoidance, n=8–12. (B) Preferences of the …
Figure 1—figure supplement 1
Requirements for Irs for preferring low salt-containing food.
(A) Binary food choice assays to assess low salt attraction by comparing Ir mutants with the control (w1118). n=8–12. (B) Tip recordings were performed on S3, S7, L3, and L4 sensilla. Shown are …
Figure 1—figure supplement 2
Gene structure of the Ir60b locus, generation of Ir60b3 and behavioral defect of Ir60b1 in high salt avoidance.
(A) Schematic of the Ir60b locus and Ir60b3 allele. The Ir60b coding exon is indicated by the blue rectangle. Ir60b3 was generated by ends-out homologous recombination by removing 768 base pairs as …
Figure 2 with 2 supplements
Contributions of different classes of gustatory receptor neurons (GRNs) to high salt avoidance.
(A) Schematic showing the names of sensilla bristles on the labellum Weiss et al., 2011. (B) Tip recordings conducted on S3, S7, and L3 sensilla using 300 mM NaCl and the indicated flies. n=10–16. (C…
Figure 2—figure supplement 1
Assaying action potentials induced by different labellar bristles in response to 300 mM salt using tip recordings.
(A) Tip recordings were performed on S-type, I-type, and L-type sensilla from control flies. n=12–14. (B) Tip recordings conducted on S3, S7, and L3 sensilla from the indicated Ir25a2 and Ir76b1 …
Figure 2—figure supplement 2
Two-way solid food choice assay to assess whether the Gr2aGAL4 mutant exhibits a deficit in avoidance of high salt.
The flies were given a choice between 1 mM sucrose versus 5 mM sucrose plus 300 mM NaCl in alternating wells of microtiter dishes. n=8. The pairwise comparison was conducted using an unpaired …
Figure 3 with 2 supplements
Measuring volume of food intake in Ir mutants using the DrosoX system.
(A–N) Each fly was exposed to two capillaries, one of which contained 100 mM sucrose (a), and the other contained 100 mM sucrose and 300 mM NaCl (b). (O and P) Each fly was exposed to two …
Figure 3—figure supplement 1
DrosoX system and measurement of food intake using strychnine and coumarin.
(A) The DrosoX system is composed of a cassette designed to accommodate five flies and a monitor that can hold five pairs of capillaries containing different solutions. Flies are exposed to both the …
Figure 3—figure supplement 2
Two-way solid food choice assay and DrosoX binary capillary feeding assay using 100 mM sorbitol with or without 300 mM NaCl.
(A) Two-way solid food choice assay to assess whether the Ir25a2, Ir60b3, and Ir76b1 mutants exhibit a deficit in high salt avoidance. The flies were given a choice between 100 mM sorbitol and 100 …
Figure 4 with 2 supplements
GCaMP6f responses of Ir60b gustatory receptor neurons (GRNs) to NaCl and other chemicals.
(A) Relative staining of the Ir60b reporter (green, anti-GFP) and the Ir76b reporter (red; anti-dsRed) in the labral sense organ (LSO) of UAS-mCD8::GFP/Ir76b-QF2;Ir60b-GAL4/QUAS-tdTomato flies. …
Figure 4—figure supplement 1
Testing whether ectopic expression of Ir60b confers responses to 300 mM NaCl, measurement of intake of sucrose plus 300 mM NaBr using the DrosoX assay, and Ca2+ response of Ir60b gustatory receptor neurons (GRNs).
(A) Tip recordings in response to 300 mM NaCl were performed on I5 and I9 sensilla of the indicated flies. UAS-Ir60b was expressed in Class B GRNs under control of the Gr33a-GAL4. n=10. (B and C) …
Figure 4—figure supplement 2
GCaMP6f responses evoked by 300 mM NaCl in Ir60b gustatory receptor neurons (GRNs) of control and Ir7cGAL4 flies, and relative expression of Ir60b and Ir7c reporters in the labral sense organ (LSO).
(A) GCaMP6f responses were stimulated by 300 mM NaCl in Ir60b GRNs from the Ir7cGAL4 mutant (Ir7cGAL4;UAS-GCaMP6f/+;Ir60b-GAL4/+) and from the Ir7cGAL4/+control (Ir7cGAL4/+;UAS-GCaMP6f/+;Ir60b-GAL4/+…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse monoclonal anti-GFP | Molecular Probes | Cat # A11120; RPID: AB_221568 | IHC (1:1000) |
| Antibody | Rabbit polyclonal anti-DsRed | Clontech | Cat # 632496; RPID: AB_10013483 | IHC (1:1000) |
| Antibody | Goat polyclonal anti-mouse Alexa Fluro 488 | Invitrogen | Cat # A32723; RRID: AB_2633275 | IHC (1:200) |
| Antibody | Goat polyclonal anti-rabbit Alexa Fluor 568 | Invitrogen | Cat # A11011; RPID: AB_143157 | IHC (1:200) |
| Chemical compound | Sucrose | Sigma-Aldrich | Cat # 9378S | |
| Chemical compound | Tricholine citrate | Sigma-Aldrich | Cat # T0252 | |
| Chemical compound | Sulforhodamine B | Sigma-Aldrich | Cat # 230162 | |
| Chemical compound | Capsaicin | Sigma-Aldrich | Cat # M2028 | |
| Chemical compound | Caffeine | Sigma-Aldrich | Cat # C02750 | |
| Chemical compound | CaCl2 dihydrate | Sigma-Aldrich | Cat # C3881 | |
| Chemical compound | KCl | Sigma-Aldrich | Cat # P9541 | |
| Chemical compound | Quinine | Sigma-Aldrich | Cat # Q1125 | |
| Chemical compound | Strychnine | Sigma-Aldrich | Cat # S8753 | |
| Chemical compound | Lobeline | Sigma-Aldrich | Cat # 141879 | |
| Chemical compound | Denatonium | Sigma-Aldrich | Cat # D5765 | |
| Chemical compound | Coumarin | Sigma-Aldrich | Cat # C4261 | |
| Chemical compound | Brilliant blue FCF | Wako Pure Chemical Industry | Cat # 027-12842 | |
| Chemical compound | Paraformaldehyde | Electron Microscopy Sciences | Cat # 15710 | |
| Chemical compound | NaCl | LPS Solution | Cat # NACL01 | |
| Chemical compound | MgCl2 hexahydrate | SAMCHUN | Cat # M0038 | |
| Chemical compound | NaBr | DUKSAN | Cat # S2531 | |
| Chemical compound | Goat serum, New Zealand origin | Gibco | Cat # 16210064 | |
| Genetic reagent (Drosophila melanogaster) | w1118 | Bloomington Drosophila Stock Center (BDSC) | BDSC:5905 | |
| Genetic reagent (Drosophila melanogaster) | Ir7a1 | Dr. Y Lee Rimal et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | Ir7g1: y1w*Mi{y+mDint2=MIC}Ir7gMI06687 | BDSC | BDSC:42420 | |
| Genetic reagent (Drosophila melanogaster) | Ir7cGAL4 | Dr. MD Gordon McDowell et al., 2022 | ||
| Genetic reagent (Drosophila melanogaster) | Ir8a1: w*TI{w[+m*]=TI}Ir8a1;Bl1L2/CyO | BDSC | BDSC:23842 | |
| Genetic reagent (Drosophila melanogaster) | Ir10a1: w1118Mi{GFPE.3xP3=ET1}Ir10aMB03273 | BDSC | BDSC:41744 | |
| Genetic reagent (Drosophila melanogaster) | Ir21a1: w1118;PBac{w+mC=PB}Ir21ac02720 | BDSC | BDSC:10975 | |
| Genetic reagent (Drosophila melanogaster) | Ir25a2 | Dr. L Vosshall Benton et al., 2009 | ||
| Genetic reagent (Drosophila melanogaster) | Ir47a1 | Dr. Y Lee Rimal et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | Ir48a1: w1118;Mi{GFPE.3xP3= ET1}Ir48aMB09217 | BDSC | BDSC:26453 | |
| Genetic reagent (Drosophila melanogaster) | Ir48b1: w1118;Mi{GFPE.3xP3= ET1}Ir48bMB02315 | BDSC | BDSC:23473 | |
| Genetic reagent (Drosophila melanogaster) | Ir51b1: w1118;PBac{w+mC=PB}rowc00387 Ir51bc00387 | BDSC | BDSC:10046 | |
| Genetic reagent (Drosophila melanogaster) | Ir52a1 | Dr. Y Lee Rimal et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | Ir52b1: w1118;Mi{GFPE.3xP3= ET1}Ir52bMB02231/SM6a | BDSC | BDSC:25212 | |
| Genetic reagent (Drosophila melanogaster) | Ir52c1: w1118;Mi{GFPE.3xP3= ET1}Ir52cMB04402 | BDSC | BDSC:24580 | |
| Genetic reagent (Drosophila melanogaster) | Ir56a1 | Dr. Y Lee Rimal et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | Ir56b1: w1118;Mi{GFPE.3xP3=ET1}Ir56bMB09950 | BDSC | BDSC:27818 | |
| Genetic reagent (Drosophila melanogaster) | Ir56d1: w*;Ir56d1 | BDSC | BDSC:81249 | |
| Genetic reagent (Drosophila melanogaster) | Ir60b1 | Dr. J Carlson Joseph et al., 2017 | ||
| Genetic reagent (Drosophila melanogaster) | Ir60b3 | Dr. Y Lee | In this study | |
| Genetic reagent (Drosophila melanogaster) | Ir62a1: y1w*;Mi{y+mDint2=MIC} Ir62aMI00895Iml1MI00895/TM3, Sb1 Ser1 | BDSC | BDSC:32713 | |
| Genetic reagent (Drosophila melanogaster) | Ir67a1: y1w*;Mi{y+mDint2=MIC}Ir67aMI11288 | BDSC | BDSC:56583 | |
| Genetic reagent (Drosophila melanogaster) | Ir75d1: w1118;Mi{GFPE.3xP3=ET1}Ir75dMB04616 | BDSC | BDSC:24205 | |
| Genetic reagent (Drosophila melanogaster) | Ir76b1 | Dr. C Montell Zhang et al., 2013 | ||
| Genetic reagent (Drosophila melanogaster) | Ir85a1: w1118;Mi{GFPE.3xP3=ET1} Ir85aMB04613 Pif1AMB04613 | BDSC | BDSC:24590 | |
| Genetic reagent (Drosophila melanogaster) | Ir92a1: w1118;Mi{GFPE.3xP3=ET1}Ir92aMB03705 | BDSC | BDSC:23638 | |
| Genetic reagent (Drosophila melanogaster) | Ir94a1 | Dr. Y Lee Rimal et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | Ir94b1:w1118;Mi{GFPE.3xP3= ET1}Ir94bMB02190 | BDSC | BDSC:23424 | |
| Genetic reagent (Drosophila melanogaster) | Ir94c1 | Dr. Y Lee Rimal et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | Ir94d1:y1w*;Mi{y+mDint2=MIC} Ir94dMI01659CG17380MI01659 | BDSC | BDSC:33132 | |
| Genetic reagent (Drosophila melanogaster) | Ir94f1: y1w*;Mi{y+mDint2= MIC}Ir94fMI00928 | BDSC | BDSC:33095 | |
| Genetic reagent (Drosophila melanogaster) | Ir94g1: w1118;Mi{GFPE.3xP3= ET1}Ir94gMB07445 | BDSC | BDSC:25551 | |
| Genetic reagent (Drosophila melanogaster) | Ir94h1 | Dr. Y Lee Rimal et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | Ir100a1: w1118;P{w+mC=EP} Ir100aG19846 CG42233G19846 | BDSC | BDSC:31853 | |
| Genetic reagent (Drosophila melanogaster) | UAS-mCD8::GFP | BDSC | BDSC:5137 | |
| Genetic reagent (Drosophila melanogaster) | UAS-mCD8::GFP | BDSC | BDSC:32184 | |
| Genetic reagent (Drosophila melanogaster) | UAS-Kir2.1 | BDSC | BDSC:6595 | |
| Genetic reagent (Drosophila melanogaster) | UAS-Ir25a | Dr. Y Lee Lee et al., 2018 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-Ir60b | Dr. Y Lee | In this study | |
| Genetic reagent (Drosophila melanogaster) | UAS-Ir76b | Dr. C Montell Zhang et al., 2013 | ||
| Genetic reagent (Drosophila melanogaster) | Ir25a-GAL4 | Dr. L Vosshall Benton et al., 2009 | ||
| Genetic reagent (Drosophila melanogaster) | Ir60b-GAL4 | Dr. C Montell Joseph et al., 2017 | ||
| Genetic reagent (Drosophila melanogaster) | Ir76b-GAL4 | Dr. C Montell Zhang et al., 2013 | ||
| Genetic reagent (Drosophila melanogaster) | ppk23-GAL4 | Dr. K Scott Thistle et al., 2012 | ||
| Genetic reagent (Drosophila melanogaster) | ppk28-GAL4 | Dr. H Amrein Cameron et al., 2010 | ||
| Genetic reagent (Drosophila melanogaster) | Gr66a-GAL4 | Dr. H Amrein Thorne et al., 2004 | ||
| Genetic reagent (Drosophila melanogaster) | Gr64f-GAL4 | Dr. A Dahanukar Lee et al., 2018 | ||
| Genetic reagent (Drosophila melanogaster) | Ir76b-QF | BDSC | BDSC:51312 | |
| Genetic reagent (Drosophila melanogaster) | QUAS-tdTomato: y1w1118; P{QUAS-mtdTomato-3xHA}26 | BDSC | BDSC:30005 | |
| Genetic reagent (Drosophila melanogaster) | Poxn∆M22-B5: y1w67c23; Mi{ET1}PoxnMB00113 | BDSC | BDSC:22701 | |
| Genetic reagent (Drosophila melanogaster) | Poxn70-28: Poxn70/CyO; twi-Gal4, UAS-2XEGFP | BDSC | BDSC:60688 | |
| Software | Origin Pro Version | Dr. Y Lee | https://www.originlab.com | |
| Software | GraphPad Prism | Dr. Y Lee | https://www.graphpd.com | |
| Software | Autospike 3.1 software | Dr. Y Lee | https://www.syntech.co.za/ | |
| Software | Fiji/ImageJ software | Dr. Y Lee | https://fiji.sc | |
| Software | ZEN lite 2.5 blue | Dr. Y Lee | https://www.zeiss.com/ |
