Proprioceptive limit detectors mediate sensorimotor control of the Drosophila leg

The datasets in this repository were used to reveal that hair plate proprioceptors on the legs of fruit flies are limit detectors that reflexively transition the direction of leg movement. We first collected a calcium imaging dataset to characterize the joint angle encoding properties of one hair plate on the fly coxa, referred to as CxHP8. We then used an electron microscopy dataset of a female ventral nerve cord (FANC) to predict the role of CxHP8 neurons in leg motor control based on their direct and indirect connectivity with motor neurons. We validated the predictions derived from the connectome by collecting and analyzing datasets where we optogenetically activated CxHP8 neurons in standing flies and silenced CxHP8 neurons in behaving flies. Additionally, we collected datasets in which flies walked on an actuated treadmill and found that chronic silencing of CxHP8 neurons lead to impaired walking kinematics and resting posture. Having supported the sensorimotor control prediction of CxHP8 neurons, we returned back to the connectome to predict the role of each hair plate on the fly leg in controlling leg movement.

Methods
Calcium imaging to characterize joint angle encoding properties

To determine the coxa joint angles in which CxHP8 neurons were active, we expressed GCaMP7f and tdTomato in them and slowly moved the platform that the front legs rested on using a 3-axis micromanipulator. We used a 2-photon microscopy setup to image the calcium activity of CxHP8 axons in the neuropil associated with the front left leg. tdTomato was used for motion correction and normalization. In addition to characterizing the joint angles encoded by CxHP8 neurons, we also determined when they are active during locomotion, grooming, and other behaviors. Analyses were done in the Jupyter notebooks, “calcium_imaging_analysis.ipynb” and “analyze_calcium_signals_during_behavior.ipynb”, found at this GitHub repository release: https://github.com/Prattbuw/Hair_Plate_Paper/releases/tag/v1.0.0. The relevant dataset is “R48A07_R20C06_GCaMP7f_tdTomato.parquet”.

Hair plate reflex circuit connectivity

Hair plate connectivity with motor neurons, either direct or indirect, was determined by using an electron microscopy dataset of a female ventral nerve cord (FANC)(Azevedo et al., 2024, Nature). Hair plate and motor neurons were identified in the dataset, along with interneurons based on their hemilineage. Hemilineages and their corresponding neurotransmitter type are found in “LacinHLTable - HL table.csv”. Connectivity analyses were done with “connectome_analysis.ipynb”, which is found here: https://github.com/Prattbuw/Hair_Plate_Paper/releases/tag/v1.0.0.

Optogenetic effects on joint kinematics and behavior

Optogenetic datasets were collected by expressing either ChrimsonR (activation) or GtACR1 (silencing) light-gated ion channels in CxHP8 neurons and using a spatiotemporally precise laser targeted at the left front leg thorax-coxa joint. In experimental trials, flies had CxHP8 neurons activated or silenced (trials were 2 seconds in duration and the laser was present for 1 second after 0.5 s from the start of the trial) while behaving on a spherical treadmill. Control trials consisted of the laser not being presented. We also collected a dataset to control for the light-effect of laser stimulation on behavior, which consisted of presenting a laser to flies that had the genetic construct GtACR1 or ChrimsonR, but not expressed in cells (i.e. R52A01 DBD > GtACR1 and R52A01 DBD > ChrimsonR). Analyses of kinematic and behavioral effects due optogenetic activation or silencing were done using “optogentics_analysis.ipynb” and “heuristic_grooming_classifier.ipynb”, which are found here: https://github.com/Prattbuw/Hair_Plate_Paper/releases/tag/v1.0.0. The relevant datasets are: “R48A07AD_R20C06DBD_ChrimsonR.pq”, “R48A07AD_R20C06DBD_GtACR1.pq”, “52A01DBD_ChrimsonR.pq” and “52A01DBD_GtACR1.pq”. Note that only data collected from the R52A01 DBD > GtACR1 flies on 4.12.24 were analyzed because their experimental conditions matched those of the flies with CxHP8 neurons silenced.

Chronic silencing of CxHP8 neurons on an actuated treadmill

The effects of chronic silencing of CxHP8 neurons on walking kinematics and resting posture were determined using an actuated treadmill system (Pratt et al., 2024, Current Biology). CxHP8 neurons were chronically silenced throughout development by expressing the inward rectifying potassium channel, Kir 2.1, within them. The walking kinematics and resting posture of CxHP8 silenced flies were compared to genetically matched control flies (i.e. R48A07 AD > Kir 2.1 and R39B11 AD > Kir 2.1). 5 highspeed cameras recorded flies walking on or resting in the treadmill system with a driving speed of 10 mm/s. DeepLabCut (Mathis et al., 2018) and Anipose (Karashchuk et al., 2021) were used to reconstruct the 3D body and leg kinematics of flies. Kinematic analyses were conducted using “treadmill_analysis.ipynb” and the kinematic results were visualized using “treadmill_visualization.ipynb”. The code is located at the following GitHub repository: https://github.com/Prattbuw/Hair_Plate_Paper/releases/tag/v1.0.0. The corresponding datasets are: “R48A07_R20C06_kir_treadmill_dataset.csv”, “R48A07AD_kir_treadmill_dataset.csv”, and “R39B11_kir_treadmill_dataset.csv”.