DOI: 10.5061/dryad.kwh70rzhq
title: A pair of dopaminergic neurons DAN-c1 mediate Drosophila larval aversive olfactory learning through D2-like receptors

description: 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.
description: # A pair of dopaminergic neurons DAN-c1 mediate Drosophila larval aversive
 olfactory learning through D2-like receptors Dataset DOI:
 [10.5061/dryad.kwh70rzhq](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