A pair of dopaminergic neurons DAN-c1 mediate Drosophila larval aversive olfactory learning through D2-like receptors
Data files
Sep 01, 2025 version files 17.98 GB
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Qi_et_al_2025_07_21.zip
17.98 GB
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README.md
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Abstract
The intricate relationship between the dopaminergic system and olfactory associative learning in Drosophila has been an intense scientific inquiry. Leveraging the formidable genetic tools, we conducted a screening of 57 dopaminergic drivers, leading to the discovery of the DAN-c1 driver, uniquely targeting a pair of dopaminergic neurons (DAN) in the larval brain. While the involvement of excitatory D1-like receptors is well-established, the role of D2-like receptors (D2Rs) remains underexplored. Our investigation reveals the expression of D2Rs in both DANs and the mushroom body (MB) of third instar larval brains. Silencing D2Rs in DAN-c1 via microRNA disrupts aversive learning, further supported by optogenetic activation of DAN-c1 during training, affirming the inhibitory role of D2R autoreceptor. Intriguingly, D2R knockdown in the MB impairs both appetitive and aversive learning. These findings elucidate the distinct contributions of D2Rs in diverse brain structures, providing novel insights into the molecular mechanisms governing associative learning in Drosophila larvae.
Dataset DOI: 10.5061/dryad.kwh70rzhq
Description of the data and file structure
The dataset includes all the immunofluorescence images, and the learning assay data related to each figure.
Files and variables
File: Qi_et_al_2025_07_21.zip
Description:
The dataset is organized based on relationships with each figure, and all data are documented under the corresponding file titles. The file names are based on the genotype of the samples, experiments, antibodies and channels information, confocal objective lens magnification times, batch numbers and sample numbers.
1. Figure 1
Data from distinct driver strains used were organized in different folders. Following are the strain names:
① TH-GAL4
② MB065B
③ R76F02AD; R55C10DBD
④ MB296B
⑤ SS1716
⑥ R58E02
⑦ R30G08
⑧ SS1696
⑨ SS0864
⑩ SS1757
For each strain, both (1) GRASP and (2) Labeling patterns are presented
(1) GRASP data are show in z -stack form (tif format)
Different batches are in different folders with numbers in the title. The green channel represents the GRASP signals, and RFP marks the morphology of the MB.
In file names, MSantiGFP is short for mouse host antiGFP antibody, MB-RFP is short for mushroom body labeled by RFP.
(2) Labeling patterns after maximum intensity projection (tif format)
Different batches are in different folders with numbers in the title. Neurons under the drivers are labeled by GFP (green), the mushroom body (MB) is labeled by RFP (red), and DANs are marked by tyrosine hydroxylase (TH) antibody (blue).
In file names, MSantiTH is short for mouse host antiTH antibody, RBantiGFP is short for rabbit host antiGFP antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
2. Figure 1-figure supplement 1
Data from distinct driver strains used were organized in different folders. Following are the strain names:
(1) TH-GAL4
(2) MB065B
(3) R76F02AD; R55C10DBD
(4) MB296B
(5) SS1716
(6) R58E02
(7) R30G08
(8) SS1696
(9) SS0864
(10) SS1757
(11) R72C04
(12) R72C08
(13) R72D03
(14) 201Y-GAL4
(15) MB247-LexA
For each strain, labeling patterns are presented patterns after maximum intensity projection (tif format)
Different batches are in different folders with numbers in the title. Representative pictures show the patterns of distinct GAL4 driver strains crossed with a UAS-GFP strain (green), except MB247-LexA is labeled with RFP (red). Blue channel represents neuropils marked by nc82 antibody.
In file names, RBantiGFP is short for rabbit host antiGFP antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
3. Figure 1-figure supplement 2
Data from distinct strains used were organized in different folders. Following are the strain names:
(1) Control straining for spGFP11 driven by MB247 driver
(2) Control straining for spGFP1-10 driven by TH-GAL4 driver
GRASP data are show in z -stack form (tif format)
Different batches are in different folders with numbers in the title. The green channel represents the GRASP signals, and RFP marks the morphology of the MB, the blue channel represents the rabbit antiGFP signals.
In file names, MSantiGFP is short for mouse host antiGFP antibody (green), MB-RFP is short for mushroom body labeled by RFP (red), RBantiGFP is short for rabbit host antiGFP antibody.
4. Figure 2
Including data (1) images of DAN-c1, and (2) behavioral data for thermogenetics
(1) Images labeling dendrites and axons of DAN-c1 after maximum intensity projection (tif format)
Dendrites and axons of DAN-c1 are labeled by DenMark (red) and sytGFP (green), correspondingly. Blue channel represents neuropils marked by nc82 antibody.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSanti nc82 is short for mouse host anti nc82 antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
(2) Thermogenetic learning data manipulating DAN-c1 in GraphPad Prism Project format.
Data from five strains under three gustatory conditions and two temperature conditions were listed (6 groups for each strain):
Strains:
① DAN-c1 x shibire
② DAN-c1
③ shibire
④ TrpA1
⑤ Dan-c1 x TrpA1
Gustatory conditions:
① QUI (quinine solution)
② DW (distilled water)
③ SUC (sucrose solution)
Temperature conditions
① 22°C
② 34°C
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
5. Figure 3
Data of D2R expression in following neuron clusters/brain structures are shown:
(1) DL1
(2) DL2a
(3) DL2b
(4) DM1a
(5) DM1b
(6) pPAM MB lobe
Data are shown in z -stack form (tif format). Different batches are in different folders with numbers in the title. For the dopaminergic neuron clusters, D2Rs are labeled by GFP (green), and DANs are marked by tyrosine hydroxylase (TH) antibody (blue).
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSantiTH is short for mouse host antiTH antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
(7) MB soma
(8) MB lobe
Data are show in z -stack form (tif format). Different batches are in different folders with numbers in the title. For the mushroom body, D2Rs are labeled by GFP (green), the mushroom body (MB) is labeled by RFP (red). In file names, RBantiGFP is short for rabbit host antiGFP antibody.
6. Figure 3-figure supplement 1
Data from distinct strains used were organized in different folders,
supporting D2R expression pattern with following D2R-GAL driver strains:
(1) R72C04
(2) R72C08
(3) R72D03
Data are shown in z -stack form (tif format). Different batches are in different folders with numbers in the title. The green channel represents GFP signals, red represents mCherry signals under D2R-GAL4 drivers, and blue marks DANs with TH antibodies.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSantiTH is short for mouse host antiTH antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
7. Figure 4
Including data
(1) Images showing D2R expression in DAN-c1.
Data are shown in z -stack form (tif format). Different batches are in different folders with numbers in the title. The green channel represents GFP signals, red represents mCherry signals under D2R-GAL4 drivers, and blue marks DANs with TH antibodies.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSantiTH is short for mouse host antiTH antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
(2) Images showing D2R knockdown in dopaminergic neurons with D2R-miR strain.
Data are shown in images after maximum intensity projection (tif format). Data from control (CR) and D2 knockdown (D2 KD) groups are separated. The green channel represents GFP signals, red represents mCherry signals under TH-GAL4 drivers, and blue marks DANs with TH antibodies.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSantiTH is short for mouse host antiTH antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel. DM1 indicates images for DM1 cluster, while DM2 represents images from pPAM cluster.
(3) Learning assay data for D2R knockdown in DAN-c1 in GraphPad Prism Project format
Data from four strains under 5 assays are provided:
Four strains
① DAN-c1 x D2R-miR
② Wild type (WT)
③ DAN-c1 x WT
④ UAS-D2R-miR
Five assays
① Olfactory associative learning
Leaning assays are under three conditions, QUI (quinine solution), DW (distilled water), and SUC (sucrose solution)
② Naïve olfactory assay to pentyl acetate (PA)
③ Naïve gustatory assay to quinine (QUI), aver
④ Naïve gustatory assay to sucrose (SUC), appe
⑤ Locomotion test
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
8. Figure 4-figure supplement 1
(1) Images showing D2R expression in DAN-d1
Data are shown in z -stack form (tif format). Different batches are in different folders with numbers in the title. The green channel represents GFP signals, red represents mCherry signals under D2R-GAL4 drivers, and blue marks DANs with TH antibodies.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSantiTH is short for mouse host antiTH antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
(2) Images labeling dendrites and axons of DAN-d1
Data are shown in images after maximum intensity projection (tif format)
Dendrites and axons of DAN-c1 are labeled by DenMark (red) and sytGFP (green), correspondingly. Blue channel represents neuropils marked by nc82 antibody.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSanti nc82 is short for mouse host anti nc82 antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
(3) Images showing D2R expression in DAN-g1
Data are shown in z -stack form (tif format). Different batches are in different folders with numbers in the title. The green channel represents GFP signals, red represents mCherry signals under D2R-GAL4 drivers, and blue marks DANs with TH antibodies.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSantiTH is short for mouse host antiTH antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
(4) Images labeling dendrites and axons of DAN-g1
Data are shown in images after maximum intensity projection (tif format)
Dendrites and axons of DAN-c1 are labeled by DenMark (red) and sytGFP (green), correspondingly. Blue channel represents neuropils marked by nc82 antibody.
In file names, RBantiGFP is short for rabbit host antiGFP antibody, MSanti nc82 is short for mouse host anti nc82 antibody, Rat anti mCherry is short for rat host anti mCherry antibody. FarRed indicates secondary antibody conjugated with Alexa 633, which is in blue channel.
(5) Learning assay data for D2R knockdown in DAN-d1 or DAN-g1 in GraphPad Prism Project format
Data from seven strains under 3 conditions are provided:
① UAS-D2R-miR
② TH-GAL4
③ TH x D2R-miR
④ DAN-d1
⑤ DAN-d1 x D2R-miR
⑥ DAN-g1
⑦ DAN-g1 x D2R-miR
Leaning assays are under three conditions, QUI (quinine solution), DW (distilled water), and SUC (sucrose solution).
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
9. Figure 4-figure supplement 2
(1) Learning assay data for D2R knockdown in dopaminergic neurons with odorant propionic acid are provided in GraphPad Prism Project format.
Data from ten strains are provided:
① WT
② UAS-D2R-miR
③ TH-GAL4
④ TH x D2R-miR
⑤ DAN-c1
⑥ DAN-d1 x D2R-miR
⑦ DAN-d1
⑧ DAN-d1 x D2R-miR
⑨ DAN-g1
⑩ DAN-g1 x D2R-miR
Odorant propionic acid (P-A) was used.
(2) D2R knockdown in dopaminergic neurons with D2R-RNAi are provided in GraphPad Prism Project format.
Data from seven strains are provided:
① UAS-RNAi
② DAN-c1 x WT
③ DAN-c1 x UAS-RNAi
④ DAN-d1
⑤ DAN-d1 x UAS-RNAi
⑥ DAN-g1
⑦ DAN-g1 x UAS-RNAi
UAS-RNAi was used to knock down D2Rs.
Leaning assays are under three conditions, QUI (quinine solution), DW (distilled water), and SUC (sucrose solution).
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
10. Figure 4-figure supplement 3
Data for naïve olfactory, naïve gustatory to quinine, naïve gustatory to sucrose, and locomotion data for all strains are provided in GraphPad Prism Project format.
Data are arranged in nine assays:
(1) Naïve olfactory assays to odorant pentyl acetate (PA) for larvae with D2R knockdown using UAS-D2R-miR
(2) Naïve olfactory assays to odorant propionic acid (P-A) for larvae with D2R knockdown using UAS-D2R-miR
(3) Naïve olfactory assays to odorant pentyl acetate (PA) for larvae with D2R knockdown using UAS-RNAi
(4) Naïve gustatory assays to quinine (QUI) for larvae with D2R knockdown using UAS-D2R-miR
(5) Naïve gustatory assays to sucrose (SUC) for larvae with D2R knockdown using UAS-D2R-miR
(6) Naïve gustatory assays to quinine (QUI) for larvae with D2R knockdown using UAS-RNAi
(7) Naïve gustatory assays to sucrose (SUC) for larvae with D2R knockdown using UAS-RNAi
(8) Locomotion tests for larvae with D2R knockdown using UAS-D2R-miR
(9) Locomotion tests for larvae with D2R knockdown using UAS-RNAi
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
11. Figure 5
Learning assay data for optogenetic manipulation in DAN-c1 in GraphPad Prism Project format.
The genotype of strain used in this experiment is DAN-c1 x ChR2. Leaning assays are under three gustatory conditions, QUI (quinine solution), DW (distilled water), and SUC (sucrose solution). Leaning assays are under four light stages, no blue light (BL), blue light during resting (resting), blue light during training (training), blue light during testing (testing). Leaning assays are under two food conditions, no ATR (all trans-retinol), and 1mM ATR.
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
12. Figure 5-figure supplement 1
Learning assay data for optogenetic manipulation in dopaminergic neurons in GraphPad Prism Project format.
The genotype of strain used in this experiment is TH-GAL4 x ChR2. Leaning assays are under three gustatory conditions, QUI (quinine solution), DW (distilled water), and SUC (sucrose solution). Leaning assays are under two light stages, no blue light (BL), and blue light during training (training). Leaning assays are under two food conditions, no ATR (all trans-retinol), and 1mM ATR.
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
13. Figure 6
(1) Learning assay data for D2R knockdown in mushroom body neurons in GraphPad Prism Project format.
Three strains were used:
① 201Y
② UAS-D2R-miR
③ 201Y x UAS-D2R-miR
Leaning assays are under three gustatory conditions, QUI (quinine solution), DW (distilled water), and SUC (sucrose solution).
(2) Optogenetic manipulation in mushroom body neurons in GraphPad Prism Project format
Learning assay data for optogenetic manipulation in DAN-c1 in GraphPad Prism Project format.
The genotype of strain used in this experiment is 201Y x ChR2. Leaning assays are under three gustatory conditions, QUI (quinine solution), DW (distilled water), and SUC (sucrose solution). Leaning assays are under four light stages, no blue light (BL), blue light during resting (resting), blue light during training (training), blue light during testing (testing). Leaning assays are under two food conditions, no ATR (all trans-retinol), and 1mM ATR.
Each individual R.I. (response index) values, statistical data, and column graphs are collected.
Access information
Other publicly accessible locations of the data:
- NA
Data was derived from the following sources:
- NA