Data and code from: Motor neurons integrate cholinergic inputs through spatial organization of diverse nicotinic receptors
Data files
Jun 11, 2026 version files 315.02 GB
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Figure1.zip
53.84 MB
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Figure2.zip
3.04 GB
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Figure3.zip
43.34 GB
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Figure4.zip
25.82 GB
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Figure5.zip
228.28 GB
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README.md
11.92 KB
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Supplementary_Figure1.zip
2.80 GB
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Supplementary_Figure2.zip
11.58 GB
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Supplementary_Figure3.zip
112.52 MB
Abstract
Neural circuit function depends not only on synaptic connectivity but also on the molecular composition and subcellular organization of neurotransmitter receptors. Here, we examine the expression, localization, and functional relevance of nicotinic acetylcholine receptors (nAChRs), the primary mediators of fast excitatory transmission in the Drosophila central nervous system. Functional nAChRs are pentamers assembled from ten subunits (α1–α7, β1–β3), yet how this diversity is deployed within defined circuits remains poorly understood.
Using T2A-Gal4 reporters and endogenous protein tagging, we identify eight nAChR subunits (α1–α3, α5–α7, β1, β2) expressed in larval motor neurons (MNs). MN-specific knockdown of individual subunits produces impairments in crawling, peristaltic timing, and protopodium dynamics, demonstrating that multiple nAChR subtypes contribute to motor output. Co-localization analyses reveal a wide range of spatial relationships, identifying subunit pairs with high, intermediate, and low overlap within MN dendritic and postsynaptic domains. Across subunit combinations, spatial organization correlates with pair-specific functional interactions: spatially segregated pairs tend to produce stronger locomotor defects when knocked down together, suggesting largely non-redundant contributions to cholinergic excitation of MNs, whereas highly co-localized pairs often show limited additional impairment. Notably, some co-localized pairs also exhibit additive effects, indicating that spatial proximity alone does not fully predict functional interaction. Dual knockdown of selected subunit pairs also reduces muscle contraction amplitude, linking receptor organization to motor output at the effector level.
Together, these results indicate that motor neurons deploy multiple nAChR populations whose spatial arrangement shapes how cholinergic inputs contribute to locomotor output.
Dataset description
This dataset contains the raw data, processed data, and analysis code used to generate the results reported in the manuscript:
Motor neurons integrate cholinergic inputs through the spatial organization of diverse nicotinic receptors
This study investigates the expression, localization, spatial organization, and functional roles of nicotinic acetylcholine receptor (nAChR) subunits in Drosophila larval motor neurons. The dataset includes confocal microscopy images used to visualize receptor expression and localization, behavioral videos used to quantify locomotion, calcium imaging recordings, muscle contraction measurements, processed measurement files, and analysis scripts used to generate the figures and statistical analyses presented in the manuscript.
The dataset is organized by manuscript figure number. Each figure directory contains the raw images, behavioral videos, processed measurements, and/or analysis code associated with the corresponding figure.
Dataset Contents
This dataset contains:
- Confocal microscopy image stacks used to examine nAChR subunit expression, endogenous tagging, conditional tagging, and receptor co-localization.
- Behavioral video recordings used to quantify larval crawling, peristalsis efficiency, peristalsis duration, protopodium folding, stride length, and locomotor phenotypes.
- Calcium imaging recordings used to quantify muscle activation during locomotion.
- Muscle contraction measurements extracted from imaging experiments.
- Processed CSV files containing measurements extracted from image and video analyses.
- R scripts used for statistical analyses and figure generation.
- Python/Jupyter notebook files used for receptor co-localization analyses and calcium imaging analyses.
Raw Data
Raw microscopy data are provided as original confocal image stacks.
Raw behavioral data are provided as original video recordings of larval locomotion.
Processed CSV files contain measurements extracted from the raw images and videos and were used for statistical analysis and figure generation.
Directory Structure and File Descriptions
Figure1
Figure 1 examines the expression of nAChR subunits in identified larval motor neurons.
Figure_1F_nAChR_T2A_Gal4_Images.zip
Contains confocal microscopy image stacks showing expression of nAChR-T2A-Gal4 reporter lines (α1–α3, α5–α7, and β2) in larval motor neurons. These images were used to identify nAChR subunits expressed in the larval motor system and to generate Figure 1F.
Figure2
Figure 2 examines endogenous and conditional tagging of nAChR subunits in identified motor neurons and their localization relative to synaptic regions.
Figure_2B_Images.zip
Contains confocal microscopy image stacks showing endogenously tagged nAChR subunits in MN1 motor neurons. These images were used to confirm expression of endogenous nAChR proteins within MN1 dendrites and to generate Figure 2B.
Figure_2D_Conditional_Tagging_Images.zip
Contains confocal microscopy image stacks showing conditional tagging of endogenous nAChR subunits in genetically identified motor neurons. These images were used to visualize receptor localization within identified motor neurons and to generate Figure 2D.
Figure_2E_Conditional_Tagging_Images.zip
Contains high-resolution confocal microscopy image stacks showing localization of conditionally tagged nAChR puncta relative to Bruchpilot-positive presynaptic active zones. These images were used to assess synaptic localization of receptor puncta and to generate Figure 2E.
Figure3
Figure 3 examines locomotor phenotypes following motor-neuron-specific RNAi knockdown of individual nAChR subunits.
Figure_3B
Contains data used to generate Figure 3B.
Subdirectories:
Alpha1_RNAi through Beta3_RNAi
Contain raw larval crawling videos for the indicated nAChR subunit RNAi knockdown genotype.
OK6-Gal4;UAS-Dicer/+
Contains raw crawling videos from genetic control larvae.
OK6-Gal4;UAS-mCherry-RNAi
Contains raw crawling videos from RNAi control larvae.
Files:
Figure_3B_OK6-Gal4>UAS-RNAi.csv
Contains processed crawling measurements used for statistical analysis and generation of Figure 3B.
OK6-Gal4>RNAi_Code.R
Contains the R code used to analyze crawling measurements and generate Figure 3B.
Figure_3C
Contains data used to generate Figure 3C.
Subdirectories:
Alpha1 through Beta3
Contain raw larval crawling videos for the indicated RNAi genotype.
CQ-Gal4;27E09-Gal4/+
Contains raw crawling videos from genetic control larvae.
Files:
Figure_3C_CQ-Gal4,27E09-Gal4>UAS-RNAi.csv
Contains processed crawling measurements used for statistical analysis and generation of Figure 3C.
CQ-Gal4,27E09-Gal4>UAS-RNAi_Code.R
Contains the R code used to analyze crawling measurements and generate Figure 3C.
Figure4
Figure 4 examines the effects of nAChR knockdown on crawling mechanics, including peristalsis efficiency, peristalsis duration, protopodium folding, and normalized stride length.
Figure_4C_AND_4D
Contains data used to generate Figure 4C and Figure 4D.
Subdirectories:
Alpha1 through Beta3
Contain raw larval crawling videos used for peristalsis analysis.
OK6-Gal4;UAS-Dicer/+
Contains control videos used for comparison.
Files:
Figure_4C_and_4D.csv
Contains processed peristalsis measurements used to quantify peristalsis efficiency and peristalsis duration.
Figure_4C_and_4D_Code.R
Contains the R code used to analyze peristalsis measurements and generate Figure 4C and Figure 4D.
Figure_4E_AND_4F
Contains data used to generate Figure 4E and Figure 4F.
Subdirectories:
Alpha1 through Beta3
Contain raw larval crawling videos used for protopodium movement analysis.
Files:
Figure_4E_and_4F.csv
Contains processed protopodium folding and displacement measurements used to quantify crawling mechanics.
Figure_4E_and_4F_Code.R
Contains the R code used to analyze protopodium measurements and generate Figure 4E and Figure 4F.
Figure5
Figure 5 examines receptor co-localization, receptor spatial organization, double RNAi locomotor phenotypes, muscle contraction, and muscle calcium activity.
Files ending in "_Images.zip" contain confocal microscopy image stacks used for receptor localization and co-localization analyses.
Files ending in "Double_RNAi_Knockdown_Videos.zip" contain larval crawling videos used to quantify locomotor behavior following simultaneous knockdown of the receptor pair indicated in the file name.
Figure_5B_Positive_Control_Images.zip
Contains confocal microscopy image stacks used as the positive control for receptor co-localization analysis (α6::HA and α6::OLLAS). These images were used to validate co-localization analysis parameters and generate Figure 5B.
Figure_5C_Negative_Control_Images.zip
Contains confocal microscopy image stacks used as the negative control for receptor co-localization analysis (Rdl::HA and α5::OLLAS). These images were used to validate co-localization analysis specificity and generate Figure 5C.
Figure_5D through Figure_5T image ZIP files
Contain confocal microscopy image stacks used to quantify co-localization between the receptor subunit pairs indicated in the file names. These datasets were used to generate the receptor co-localization analyses shown in Figure 5D through Figure 5T.
Figure_5L through Figure_5T double RNAi video ZIP files
Contain raw larval crawling videos used to quantify locomotor behavior following simultaneous motor-neuron-specific knockdown of the receptor pairs indicated in the file names. These datasets were used to generate the locomotor analyses shown in Figure 5L through Figure 5T.
Figure_5_Double_RNAi_code.R
Contains the R code used to analyze all double RNAi locomotor datasets presented in Figure 5.
Figure_5U.zip
Contains muscle contraction imaging data, processed measurements, and analysis files used to quantify muscle contraction amplitude during larval locomotion. These data were used to generate Figure 5U.
Figure_5V.zip
Contains calcium imaging data, processed measurements, and analysis files used to quantify GCaMP6s fluorescence responses during larval locomotion. These data were used to generate Figure 5V.
Figure_5U_5V_code.ipynb
Contains the Jupyter notebook used to analyze muscle contraction and calcium imaging datasets and generate Figure 5U and Figure 5V.
Colocalization_code 7.31.27 PM copy.ipynb
Contains the Jupyter notebook used to analyze receptor co-localization datasets and generate co-localization plots presented in Figure 5.
Supplementary_Figure1
Contains validation and conditional-tagging microscopy datasets.
CQ-Gal4;UAS-FLP/Alpha3-ALFA.zip
Contains confocal microscopy image stacks demonstrating conditional tagging of α3-ALFA in CQ-positive motor neurons.
CQ-Gal4;UAS-FLP/Alpha7-OLLAS.zip
Contains confocal microscopy image stacks demonstrating conditional tagging of α7-OLLAS in CQ-positive motor neurons.
RRa-Gal4;UAS-myr-GFP/Alpha1-OLLAS.zip
Contains confocal microscopy image stacks demonstrating MN1-specific conditional tagging of α1-OLLAS.
Supplementary_Figure2
Supplementary_Figure2_Videos.zip
Contains representative larval crawling videos corresponding to locomotor phenotypes presented in Supplementary Figure 2.
Supplementary_Figure3
Movie S4.mp4.zip
Contains representative muscle calcium imaging recordings during larval locomotion. These videos illustrate the muscle contraction and calcium imaging analyses used throughout the study.
Variable Definitions
- Gene: nAChR subunit analyzed.
- Type: Experimental condition.
- Genotype: Full genotype of the larvae analyzed.
- Track: Tracking identifier for a larva or behavioral measurement.
- Length: Larval crawling length measured in pixels.
- Duration: Duration of one peristaltic wave measured in seconds.
- Peristalsis per cm: Number of peristaltic waves per centimeter traveled.
- Efficiency: Distance traveled per peristaltic wave.
- Frames per peristalsis: Number of video frames corresponding to one peristaltic wave.
- Folding: Raw protopodium folding measurement.
- Percentf: Percent folding or percent muscle contraction measurement.
- Displacement: Raw displacement measurement.
- Percentd: Percent displacement measurement.
- Muscle contraction: Raw muscle contraction measurement.
- DF/F: Change in fluorescence normalized to baseline fluorescence.
- RNAi: RNA interference construct used to reduce expression of the indicated gene.
- OK6-Gal4: Broad motor neuron driver.
- CQ-Gal4;27E09-Gal4: Restricted motor neuron driver.
- D42-mCherry: Motor neuron marker used during co-localization analyses.
- HA, OLLAS, ALFA, GFP: Protein tags used to label receptor subunits.
- Rdl: GABA-gated chloride channel used as a negative control for co-localization experiments.
- GCaMP6s: Genetically encoded calcium indicator used for calcium imaging experiments.
Software and analysis
Statistical analyses were conducted using R and Python/Jupyter notebooks.
Required R packages:
ggplot2, dunn.test, ggsignif, dplyr, tidyr, ggpubr, rstatix, FSA, ggbeeswarm, palmerpenguins, ggforce, and magick.
Required Python packages:
pandas, numpy, matplotlib, seaborn, scipy, and scikit-posthocs.
Additional software:
- Fiji/ImageJ
- wrMTrck ImageJ plugin
- Imaris 10.0.1
- MATLAB
Some analysis scripts contain computer-specific file paths. To rerun analyses, users should place the required files in the same directory as the script or update file paths accordingly.
System Files
Files such as .DS_Store and files beginning with . are automatically generated by macOS. These files do not contain experimental data, images, videos, measurements, code, or analysis results and may be safely ignored.