Aminergic and peptidergic modulation of insulin-producing cells in Drosophila
- Ache Lab, Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, Julius-Maximilians-University of Würzburg, Am Hubland, Germany
- Zandawala Lab, Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, Julius-Maximilians-University of Würzburg, Am Hubland, Germany
- Department of Biochemistry and Molecular Biology, University of Nevada Reno, United States
Figures
Figure 1 with 5 supplements
Expression of neuromodulator receptors in insulin-producing cells (IPCs).
(A) Expression of biogenic amine and neuropeptide receptors in IPC single-nucleus transcriptomes. Novel receptors are depicted in black, and receptors previously shown to be expressed in IPCs are …
Figure 1—figure supplement 1
Insulin-producing cells (IPCs) express receptors for biogenic amines, neuropeptides, and classical neurotransmitters.
(A) Cluster analysis of IPCs based on Ilp2, Ilp3, Ilp5, Dsk, and Piezo expression. Expression levels are depicted as a heatmap where each row represents a single-cell. Expression was scaled based on …
Figure 1—figure supplement 2
Expression of select neuromodulator receptors across individual insulin-producing cells (IPCs).
t-distributed Stochastic Neighbor Embedding (t-SNE) plots showing expression of select biogenic amine and neuropeptide receptors across IPC transcriptomes. Note that some receptors such as AstA-R1 …
Figure 1—figure supplement 3
Overview of neuromodulator receptor expression in the brain.
T2A-GAL4 knock-in lines for different receptors drive broad expression of nuclear mCherry (magenta). Insulin-producing cells (IPCs) (green) and the neuropil (blue) have been labeled using DILP2 and …
Figure 1—figure supplement 4
Neuromodulator receptors expressed in insulin-producing cells (IPCs).
Representative confocal stacks showing expression of different neuromodulator receptors (magenta) in IPCs (labeled using DILP2 antibody in green). Receptor expression was assessed by driving nuclear …
Figure 1—figure supplement 5
Neuromodulator receptors not expressed in insulin-producing cells (IPCs).
Representative confocal stacks showing a lack of expression of different neuromodulator receptors (magenta) in IPCs (labeled using DILP2 antibody in green). Receptor expression was assessed by …
Figure 2 with 2 supplements
Modulation of individual insulin-producing cells (IPCs) by aminergic and peptidergic neurons in patch-clamp recordings.
(A) Schematic of setup for patch-clamp recordings and optogenetic activation. (B) Anatomy of an example driver line labeling AstANs (gray) and antibody staining against DILP2 labeling IPCs (green). …
Figure 2—figure supplement 1
Expression of the ModN driver lines in the brain and VNC.
The expression pattern of each ModN line was visualized via GFP-expression (magenta) in the fly brain and VNC using the respective GAL4 driver line. IPCs were visualized using anti-DILP2 (green), …
Figure 2—figure supplement 2
Insulin-producing cell (IPC) responses to repeated activation of ModNs and statistical analysis of IPC responses to ModN activation for all lines.
(A) Example recording from IPC shows excitation upon dopamine (DAN) activation across ten repetitions (black traces) for 100 ms activation (left) and 1 s activation (right). Spike events are …
Figure 3 with 1 supplement
Latency analysis and singly fly recordings.
(A) Threshold-based latency analysis for excited (magenta) and inhibited (blue) insulin-producing cells (IPCs) during activation of dopamine (DANs), octopamine (OANs), neuropeptides AstA (AstANs), …
Figure 3—figure supplement 1
Cluster distribution of multiple insulin-producing cells (IPCs) recorded in the same individuals during neuropeptides AstA (AstANs) activation.
The recorded IPCs in Fly #1 (example from Figure 3) were divided into an excited, inhibited, and an unaffected cluster based on threshold clustering. In Fly #2, all IPCs were inhibited, while in Fly …
Figure 4 with 1 supplement
Modulation of the insulin-producing cell (IPC) population by aminergic and peptidergic neurons in calcium imaging experiments.
(A) Schematic of the setup for optogenetic activation during calcium imaging. (B) Example images of the GCaMP6m-expressing IPC cell bodies, delimited as ROIs during a time series before and during a …
Figure 4—figure supplement 1
Comparison of insulin-producing cell (IPC) responses to ModN activation for different durations in calcium imaging recordings.
(A) Median area under the curve (AUC) (empty circles) and maximum and minimum dF/F values (filled circles) for different activation lengths and all ModNs tested. Magenta, excited, blue, inhibited. (B…
Figure 5
Neurons containing classical transmitters provide heterogeneous input to insulin-producing cells (IPCs).
(A – D) Presynaptic neurons to IPCs in the FlyWire connectome contain acetylcholine (A), glutamate (B), GABA (C), or unknown transmitters (D). Scale bar = 100 µm. (E) Connectivity map of presynaptic …
Figure 6
Summary of neuromodulation of insulin-producing cells (IPCs).
(A) Comparison of overall activity shifts for all driver lines and IPCs tested between patch-clamp (black) and calcium imaging (white). (B) Comparison of cluster proportions between patch-clamp …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (D. melanogaster) | UAS-NLS-mCherry | Bloomington Drosophila Stock Center | RRID:BDSC_38425 | Used for receptor mapping |
| Strain (D. melanogaster) | Oct-TyrR-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_86138 | Used for receptor mapping |
| Strain (D. melanogaster) | 5HT7-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84592 | Used for receptor mapping |
| Strain (D. melanogaster) | Oct-alpha2R-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84610 | Used for receptor mapping |
| Strain (D. melanogaster) | AstAR1-T2A-GAL4 | Deng et al., 2019 | Used for receptor mapping | |
| Strain (D. melanogaster) | MsR1-(B)-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84653 | Used for receptor mapping |
| Strain (D. melanogaster) | AstAR2-(A/C)-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84594 | Used for receptor mapping |
| Strain (D. melanogaster) | sNPFR-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84691 | Used for receptor mapping |
| Strain (D. melanogaster) | CCKLR17D1-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84605 | Used for receptor mapping |
| Strain (D. melanogaster) | CrzR-T2A-GAL4 | Kondo et al., 2020 | Used for receptor mapping | |
| Strain (D. melanogaster) | Dh31R-(A/B/C)-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84625 | Used for receptor mapping |
| Strain (D. melanogaster) | Dop2R-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84628 | Used for receptor mapping |
| Strain (D. melanogaster) | LkR-GAL4 Knock-in mutant | Zandawala et al., 2018 | Used for receptor mapping | |
| Strain (D. melanogaster) | Oct-alpha2R-Trojan-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_67636 | Used for receptor mapping |
| Strain (D. melanogaster) | 5-HT1A-T2A-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84588 | Used for receptor mapping |
| Strain (D. melanogaster) | R96A08-LexA- p65-vk37::LexOp-dilp2::GFP;20x-UAS-CsChrimson- attp2/TM6b | Liessem et al., 2023 | Used for optogenetic activation during patch-clamp recordings | |
| Strain (D. melanogaster) | R96A08-LexA-p65-vk37/CyO;LexOp-GCaMP6m-vk5, 20X-UAS-CsChrimson. mVenus-su(Hw) attp1/TM6 | This study | Used for optogenetic activation during calcium imaging | |
| Strain (D. melanogaster) | TH-GAL4 | Friggi-Grelin et al., 2003 | Used for optogenetic activation | |
| Strain (D. melanogaster) | AstA-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_84593 | Used for optogenetic activation |
| Strain (D. melanogaster) | Tdc2-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_9313 | Used for optogenetic activation |
| Strain (D. melanogaster) | LK-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_51993 | Used for optogenetic activation |
| Strain (D. melanogaster) | MS-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_51986 | Used for optogenetic activation |
| Strain (D. melanogaster) | AKH-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_25684 | Used for optogenetic activation |
| Strain (D. melanogaster) | TK-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_51973 | Used for optogenetic activation |
| Strain (D. melanogaster) | sNPF-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_51991 | Used for optogenetic activation |
| Strain (D. melanogaster) | DH31-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_51988 | Used for optogenetic activation |
| Strain (D. melanogaster) | TrH-GAL4 | Yi Rao Laboratory Chinese Institute for Brain Research, Beijing (China) | CG9122 | Used for optogenetic activation |
| Strain (D. melanogaster) | GFP-p10 | Bloomington Drosophila Stock Center | RRID:BDSC_32201 | Used for patch-clamp recordings |
| Antibody | anti-RFP (primary guinea pig polyclonal) | Dr. Susan Brenner-Morton, Columbia, USA | Enhances RFP signal, diluted 1:5000 | |
| Antibody | anti-DILP2 (primary rabbit polyclonal) | Veenstra et al., 2008 | RRID:AB_2569969 | Used to label IPCs, diluted 1:2000 |
| Antibody | anti-nc82 (primary mouse monoclonal) | DSHB | RRID:AB_2314866 | Used to label neuropils, diluted 1:500 |
| Antibody | anti-GFP (primary chicken polyclonal) | Abcam | RRID:AB_2269474 | Enhances GFP signal, diluted 1:1000 |
| Antibody | Donkey anti-rabbit Alexa Fluor 647 (secondary donkey polyclonal) | ThermoFisher Scientific | RRID:AB_2536183 | Diluted 1:1000 |
| Antibody | Goat anti-guinea pig Alexa Fluor 555 (secondary goat polyclonal) | ThermoFisher Scientific | RRID:AB_2535856 | Diluted 1:1000 |
| Antibody | Donkey anti-mouse Alexa Fluor 488 (secondary donkey polyclonal) | ThermoFisher Scientific | RRID:AB_141607 | Diluted 1:1000 |
| Antibody | Goat anti-chicken Alexa Fluor 488 (secondary goat polyclonal) | ThermoFisher Scientific | RRID:AB_2534096 | Diluted 1:200 |
| Antibody | Goat anti-rabbit Alexa Fluor 555 (secondary goat polyclonal) | ThermoFisher Scientific | RRID:AB_2535849 | Diluted 1:200 |
| Antibody | Goat anti-mouse Alexa Fluor 635 (secondary goat polyclonal) | ThermoFisher Scientific | RRID:AB_2536184 | Diluted 1:400 |
| Chemical compound | SigmaCote | Sigma-Aldrich | cat. no. SL2; RRID:SCR_008988 | siliconizing reagent |
| Chemical compound | Vectashield Antifade Mounting Medium | VEC-H-1000 | Biozol | - |
| Chemical compound | all-trans-retinal | Sigma-Aldrich | R2500 | Used in optogenetic experiments |
| Software, algorithm | OCULAR | OCULAR | Image acquisition software (patch clamp) | |
| Software, algorithm | Micro-Manager 2.0 for Image J | Edelstein et al., 2014 | Image acquisition software (calcium imaging) | |
| Software, algorithm | pCLAMP 10 | Molecular Devices | Used for recordings (Optogenetic activation and patch clamp) | |
| Software, algorithm | MATLAB R2021a | The Mathworks | Used for data analysis and statistical testing | |
| Software, algorithm | Jupyter Notebook | Project Jupyter | Pre-processing of calcium imaging data |
-
BDSC = Bloomington Drosophila Stock Center.
Table 1
p-values for calcium imaging area under the curves (AUCs) before and after activation.
| Line | p-values | |||
|---|---|---|---|---|
| Global | Excited | Unaffected | inhibited | |
| AstAN | <0.0001 | <0.0001 | 0.6875 | 0.0078 |
| OAN | <0.0001 | <0.0001 | 0.9101 | <0.0001 |
| DAN | 0.0036 | <0.0001 | 0.0195 | 0.0001 |
| LKN | 0.3684 | <0.0001 | 0.0495 | <0.0001 |
| MSN | <0.0001 | 0.1562 | 0.3593 | <0.0001 |
Additional files
-
Supplementary file 1
Tables listing abbreviations used for receptors, neuropeptides, and biogenic amines (A) and G-protein prediction scores for selected receptors in insulin-producing cells (IPCs) (B).
- https://cdn.elifesciences.org/articles/99548/elife-99548-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/99548/elife-99548-mdarchecklist1-v1.docx
