Anatomical Distribution and Behavioral Contributions of 13A and 13B Hemilineages

(A) Schematic showing segmental distribution of 13A (green) and 13B (cyan) neurons across pro-, meta-, and meso-thoracic segments (T1, T2, T3) of VNC.

(B) Confocal image: Six GABAergic 13A neurons (green arrowheads) and six 13B neurons (cyan arrowheads) in each VNC hemisegment, labeled with GFP (green) driven by R35G04-GAL4-DBD, GAD-GAL4-AD. Neuropil in magenta (nc82). Panel B’ provides a zoomed-in view of T1 region.

(C) EM reconstructions: 62 13A neurons (green) and 64 13B neurons (cyan) in right T1. Ventral side up.

(D) Continuous activation of 13A and 13B neurons labeled by R35G04-GAL4-DBD, GAD-GAL4-AD in dusted flies, results in reduced front leg rubbing and head sweeps, and unusual leg extensions (N=7, total time = 4 min).

(E, E’) Silencing 13A and 13B neuron subsets (panel B) (R35G04-GAL4-DBD, GAD-GAL4-AD > UAS TNTe) locks one or both front legs in flexion (blue arrowhead) in clean (E) (n=5) and dusted (E’) (n=13) headless flies. Panel E” provides zoomed-in view of front legs with a schematic illustrating leg flexion.

(F, F’) Activating 13A and 13B neuron subsets (R35G04-GAL4-DBD, GAD-GAL4-AD > UAS CsChrimson) induces front leg extension (orange arrowhead) in both clean (F) (n=5), and dusted (F’) (n=10) headless flies. Panel F” provides zoomed-in view of front legs with a schematic illustrating leg extension. Also see Figure 1—Video 1.

Manipulating activity of six 13A neurons and six 13A neurons in headless flies.

Inactivation: Legs locked in flexion in clean and dust covered flies. Activation: legs extended in clean and dust covered flies.

Expression pattern in the central nervous system of various lines used for behavior experiments

(A) R35G04-DBD and GAD1-AD Split GAL4 combination labels approximately 6-7 13A neurons and 3 13B neurons per thoracic hemisegment. Ectopic expression is observed in a few neurons in the brain, and in the ventral nerve cord (VNC) in about 9 neurons per hemisegment (possibly 13A neurons with posterior cell bodies or 3B neurons) and 4 neurons per hemisegment (possibly 0A) within the Accessory Mesothoracic neuropil (AMNp) and T2 midline.

(B) R35G04-DBD and Dbx-AD Split GAL4 combination specifically labels a subset of 13A neurons already included in the R35G04-DBD and GAD1-AD Split GAL4 line, thereby isolating 13A neurons without labeling 13B neurons. It also labels approximately 3 neurons per hemisegment with posterior cell bodies and shows no ectopic expression in the brain.

(C) R11C07 and Dbx-AD Split GAL4 combination labels another distinct subset of 13A neurons, with no ectopic expression in the brain.

(D) R11B07-DBD and GAD1-AD Split GAL4 combination labels three 13B neurons. It occasionally labels two ectopic ascending neurons per hemisegment.

Spatial Map of Premotor 13A Neurons Correlates With Their Connections to Motor Neurons (MNs)

(A) Hierarchical Clustering of 13A Hemilineage. Clustering of 13A neuron types in the right T1 segment was performed using NBLAST, resulting in identification of 10 morphological groups or Clusters. Neurons are named based on morphological clustering. For example, all neurons in the 13A-3 cluster have similar morphology, with 10 neurons labeled as 13A-3 (a-j) (olive). Images of each 13A neuron are shown in Figure 2—Figure Supplement 1. Also see Figure 2—Video 2.

(B) Cosine similarity graph showing pairwise similarity between 13A neurons based on their MN connectivity patterns. 13A neurons are organized based on anatomical clusters obtained with NBLAST as described above. It depicts a correlation between anatomy of 13A neurons and their connections to MNs. For example, 13A-1a, −1b, −1c, −1d (cluster 1) connect to same set of MNs, therefore have high cosine similarity with each other (as seen across the diagonal). Graph also gives insights into groups of 13As that control similar muscles. For example, cluster 1 neurons have high cosine similarity with cluster 3 13A neurons (while, 3g neuron is an exception).

(C, D) Morphologies of 13A cluster and downstream MNs: Examples of 13A neuron types classified using NBLAST, alongside their downstream MNs. Neurons within same cluster have similar anatomy, closely positioned within the VNC, with dendrites and axons occupying similar spatial regions. EM reconstructions of two distinct 13A clusters in T1-R are shown. The spatial positions of 13A axons tile the leg neuropil and correspond to the dendrites of MNs they synapse onto. 13A clusters are in black, MNs: extensors (red and brown), flexors (blue). MNs with highest number of synapses are highlighted in red. A= anterior, P= posterior, D= dorsal, V= ventral. Left side depicts a ventral side up, while right side shows ventral toward the right.

Spatial Map and Connectivity of Premotor 13A Neurons

(A1-A6) Top: EM reconstruction showing morphology of individual 13A neurons in cluster-1 in the right hemisegment of prothoracic ganglion (T1) region of VNC. Bottom: Leg schematic illustrating muscles innervated by the MNs inhibited by Cluster-1 13A neurons.

(A7) Top: EM reconstruction showing morphology of all cluster-1 13A neurons combined. Bottom: Connectivity matrix showing cluster 1 13A neurons and their MN connections. These neurons connect to Sternotrochanter extensor (SE) and trochanter extensor (Tr E) both involved in trochanter extension. Edges represent synaptic weight.

(B1-B6) Top: EM reconstruction showing morphology of individual Cluster-3 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-3 13A neurons.

(B7) Top: Morphology of Cluster-3 13A neurons combined except 13A-R-3g. Bottom: Connectivity matrix showing connections between cluster-3 13A neurons and MNs. Tr =trochanter, Tr E = trochanter extensor, Tr F = trochanter flexor, Ta D= tarsus depressor.

(B8) Top: Morphology of Cluster-2 13A neuron. Bottom: Leg schematic for muscles innervated by the Cluster-2 13A neuron.

(C1-C5) Top: EM reconstruction showing morphology of individual Cluster-4 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-4 13A neurons.

(C6) Top: Morphology of Cluster-4 13A neurons combined. Bottom: Connectivity matrix showing connections between cluster-4 13A neurons and MNs.

(D1-D8) Top: EM reconstruction showing morphology of individual Cluster-5 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-5 13A neurons

(D9) Top: Morphology of Cluster-5 13A neurons combined. Bottom: Connectivity matrix showing connections between cluster-5 13A neurons and MNs. Fe R = femur reductor.

(E1-E6) Top: EM reconstruction showing morphology of individual Cluster-6 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-6 13A neurons.

(E7) Left: Morphology of Cluster-6 13A neurons combined. Right: Connectivity matrix showing connections between cluster-6 13A neurons and MNs.

(F1-F3) Top: EM reconstruction showing morphology of individual Cluster-7 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-7 13A neurons.

(F4) Top: Morphology of Cluster-7 13A neurons combined. Bottom: Connectivity matrix showing connections between cluster-7 13A neurons and MNs.

(G1-G5) Top: EM reconstruction showing morphology of individual Cluster-8 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-8 13A neurons.

(G6) Top: Morphology of Cluster-7 13A neurons combined. Bottom: Connectivity matrix showing connections between cluster-7 13A neurons and MNs.

(H1-H8) Top: EM reconstruction showing morphology of individual Cluster-9 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-9 13A neurons.

(H9) Top: Morphology of Cluster-9 13A neurons combined. Bottom: Connectivity matrix showing connections between cluster-9 13A neurons and MNs.

(I1-I8) Top: EM reconstruction showing morphology of individual Cluster-10 13A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-10 13A neurons.

(I9) Top: Morphology of Cluster-10 13A neurons combined. Bottom: Connectivity matrix showing connections between cluster-10 13A neurons and MNs.

Extensors in orange and Flexors blue.

Connectivity Matrix of 13A Neurons and the Motor Neurons Showing Left-Right Comparison and Spatial Map.

The 13A neurons that belong to the same anatomical cluster connect to same set of motor neurons (MNs). 13A neurons that have similar morphology are shown in the same color. Clusters that contain similar neurons on the right and the left are shown in one color. The edge width between 13A neurons and motor neurons corresponds to the normalized synaptic weight. Each cluster on the right and the left connects to the same set of MNs. Synaptic weights on the left15 are greater than on the right, but the connections and preferred partners are the same. The leg schematic shows the muscles that these MNs innervate. Extensor muscles: orange, flexors: blue.

Anatomical classification of 13B neurons.

(A) Hierarchical clustering of 13B hemilineage in the right (R) T1 based on NBLAST similarity scores. Each cluster is depicted in a different color. The neurons having similar morphology represent one cluster. Neurons are named based on morphological clustering, for example, all neurons in the 13B-R-1 series have similar morphology and contain 8 neurons 13B-R-1(a-h). Bottom panel shows visualization of clusters in a 2D space. Each subplot depicts a distinct cluster, with different colors indicating different clusters.

(B) Cosine similarity graph showing the pairwise similarity between 13B neurons based on their connections to all downstream neurons. 13B neurons are named based on the anatomical clusters obtained with NBLAST as described above.

Neurons Downstream of a Primary 13A-10f-α Neuron.

Motor neurons (red) constitute 58.4% of the total downstream synapses. Other downstream connections include glutamatergic hemilineages, 21A, 24B (yellow), cholinergic hemilineages 7B, 3A, 20A (green), GABAergic hemilineages 12B (gray), and other unknown neurons (blue, orange).

Percentage of downstream synapses of 13A neurons that directly connect to motor neurons (MN).

MN connections in red and other neurons in gray. (A1-A5) Cluster 1 13A neurons. (B1) Cluster 2 13A neuron. (C1-C11) Cluster 3 13A neurons. (D1-D5) Cluster 4 13A neurons. (E1-E8) Cluster 5 13A neurons. (F1-F7) Cluster 6 13A neurons. (G1-G3) Cluster 7 13A neurons. (H1-H5) Cluster 8 13A neurons. (I1-I9) Cluster 9 13A neurons. (J1-J7) Cluster 10 13A neurons.

13A neurons in the right front leg neuromere (T1R). Primary neurons in brown. Secondary neurons in green and red.

Three sub-bundles of hemi-lineage 13A neurons are shown across 2D EM sections (left) and 3D rendering (right) of the cell bodies and 13A axonal tracks entering the neuropil.

13A morphological clusters

13A cluster 6 neurons (red) and downstream Tibia extensor MNs (feti and seti, green)

Inhibitory Circuitry for Antagonistic Muscle Control

(A) Schematic of inhibitory circuit motifs (A1) Direct MN inhibition by 13A/B neurons. (A2) Flexor inhibition and extensor disinhibition: 13As-i inhibit flexor MNs and disinhibit extensor MNs by inhibiting 13As-ii. (A3) Extensor inhibition and flexor disinhibition: 13As-ii inhibit extensor MNs and disinhibit flexor MNs by inhibiting 13As-i. (A4) 13B mediated disinhibition: 13Bs disinhibit MNs by targeting premotor 13As, while some also directly inhibit antagonistic MNs. (A5) Reciprocal inhibition among 13A groups that inhibit antagonistic MNs may induce flexor-extensor alternation.

(B) Connectivity matrix: Inhibitory connections regulating antagonistic MNs of the medial joint. Leg schematic shows tibia extensor (orange) and flexor (blue) muscles, innervated by respective MNs. Flexor-inhibiting 13A neurons (13As-i) in blue, and extensor inhibiting 13As (13As-ii) in orange.

Direct MN inhibition: Primary neurons (13A-10f-α, 9d-γ, and 10e-δ) and 13A-10c (13As-i) connect to tibia flexor MNs (blue edges), making a total of 85, 219, 155, 157 synapses, respectively. Twelve secondary 13As ii inhibit tibia extensor MNs (orange edges), with strong connections from 13A-9f, −9e, and −10d totaling 188, 275, 155 synapses, respectively.

Reciprocal inhibition: Three neurons from 13As-i inhibit six from 13As ii, with 13A-10e-δ connecting to 13A-9f (19 synapses), −9e (31), and −10d (14). 13A-10c connects to 13A-8a (6), −8b (12), and −8e (5). 13A-9d-γ connects to 13A-8e (8). Conversely, three from 13As-ii inhibit two neurons from 13As i, with 13A-9f connecting to 13A-10f-α (25) and −9d-γ (6), and 13A-10d connecting to 13A-10f-α (8), −9d-γ (7), and −10e-δ (15). 13A-9e connects to 13A-10f-α (21) and −10c (47) (black edges).

Disinhibition by 13B neurons: 13B connects to 13As-i (13A-10f-α and −9d-γ) (totaling 78 and 50 synapses) (green edges), disinhibiting flexor MNs. 13B-2g and −2i also directly inhibit tibia extensor MNs.

Reciprocal inhibition for multi-joint coordination: Primary 13As (10e-δ and 10f-α) target proximal extensor MNs and medial/distal flexors, while secondary 13As (9e and 9f) target antagonist muscle groups, indicating generalist 13As coordinate muscle synergies through reciprocal inhibition.

(C) Proprioceptive feedback: Sensory feedback from proprioceptors onto reciprocally connected 13As could turn off corresponding MNs and activate antagonistic MNs. Flexion-sensing proprioceptors target extensor MNs and 13As-i that inhibit tibia flexor MNs. Thus, after flexion completion, this feedback could induce extension and inhibit flexion via 13A neurons. Extension-sensing proprioceptors target tibia flexor MNs and two 13As (13As-ii) that inhibit extensor MNs. Claw extension neurons also connect to 13A-δ. 13Bs that disinhibit flexor MNs also receive connections from extension-sensing proprioceptors. Thus, extensor-sensing neurons can initiate flexion by directly activating MNs, disinhibit flexion by activating 13As-ii/13B neurons, and inhibit extension via 13As-ii neurons. Also see Figure 3—figure supplement 3.

Disinhibition Matrix

(A) Network Visualization of all 13A and 13B Neuronal Interconnections. Network graph showing all 13B (presynaptic) to 13A (postsynaptic) connections (blue edges), 13A to 13A connections (red edges), 13A to 13B connections (gray edges) and 13B to 13B connections (green edges). 13A nodes in red. 13B nodes in blue.

(B) Disinhibition mediated by 13B neurons: A connectivity graph showing all 13B to 13A connections.The leg schematic on the right side shows targets of motor neurons inhibited by these 13A neurons, which are disinhibited by 13B neurons. Nodes of the same color within a lineage represent neurons within the same morphological cluster. Edge color corresponds to the postsynaptic 13A targets. Extensors in orange, Flexors in blue.

(C) Disinhibition mediated by 13A-13A connections: A connectivity graph highlighting connections within 13A neurons that leads to disinhibition of motor neurons. Nodes of the same color represent same 13A anatomical cluster. Primary 13A neuron nodes are highlighted in blue, except for 13AR-6a(-ε) that is highlighted in red. Color of the edges corresponding to the color of a presynaptic neuron.

(D) Disinhibition mediated by 13A-13B connections: A connectivity graph showing connections from 13A neurons to 13B neurons. Note that most of these postsynaptic 13B neurons are premotor (shown in panel F).

(E) Disinhibition mediated by 13B-13B connections: A connectivity graph showing interconnections between 13B neurons.

(F) Premotor 13B neurons: A connectivity graph showing premotor 13B neuron connections to MNs. 13B neurons that are morphologically related (same cluster) are shown in same color. 13B neurons that belong to the same cluster do not connect to same set of motor neurons. Extensors in orange, Flexors in blue.

Neurons Downstream of a 13B Neuron (13B-4i).

13A neurons (red) comprise 36.8% of the total downstream synapses. 20/22A cholinergic neurons (green), 24.5% of the total synapses. 9A neurons (cyan)(6.5%), two contralateral interneurons (9%), 13B neuron (1.9%), 3B (1.9%), ipsilateral interneurons (possibly 21A?) (11%) of the total downstream partners. A. Individual downstream neurons. B. Aggregate of the same type of neurons.

Sensory Feedback Onto Inhibitory 13A Neurons and Motor Neurons.

Connectivity Matrix Showing Position Sensing Claw Neurons and Motion Sensing Hook Neurons Send Feedback Connections to 13A Neurons and Antagonistic MNs. Flexion Position and Motion Sensing Proprioceptors (Blue) Neurons Connect to Tibia Extensor MNs and Primary 13As (13As-I Group) That Inhibit Tibia Flexor MNs. Thus, When the Flexion Is Complete, It Could Induce Extension and Inhibit Flexion Via 13A Neurons. Similarly, Extension Position and Motion Sensing Proprioceptors (Orange) Neurons Connect to Tibia Flexor MNs and Two 13As (13As-Ii Group) That Inhibit Tibia Extensor MNs. Overall, Extension Position Sensing Neurons Could Activate Flexion and Inhibit Extension. Synaptic Weights Are Shown Between Connections Also Indicated by the Edge Thickness.

13A and 13B Neurons Are Required for Leg Coordination During Grooming

(A) Intra-joint coordination and muscle synergies: Angular velocities of proximal (P, blue), and medial (M, cyan) joints predominantly move synchronously, while distal (D, purple) can move in or out of phase during leg rubbing.

(B) Neuronal labeling of 13A neurons: Confocal image showing six Dbx positive 13A neurons per hemisegment labeled by GFP using R35G04-GAL4-DBD, Dbx-GAL4-AD in VNC. Neuroglian (magenta) labels axon bundles.

(C) Neuronal labeling of 13B neurons: Confocal image showing three 13B neurons per hemisegment labeled by GFP using R11B07-GAL4-DBD, GAD-GAL4-AD. Nc82 (magenta) labels neuropil.

(D-K) Effects of neuronal activity manipulation: Activation and silencing of 13A neurons in dusted flies using R35G04-GAL4-DBD, Dbx-GAL4-AD with UAS CsChrimson or UAS Kir, respectively (n=19 activation, n=12 silencing). Control: AD-GAL4-DBD Empty Split with UAS CsChrimson or UAS Kir. For 13B neurons, R11B07-GAL4-DBD, GAD-GAL4-AD with UAS CsChrimson or UAS GtACR1, respectively (n=9 activation, n=7 silencing); control: AD-GAL4-DBD Empty Split with UAS CsChrimson or UAS GtACR1. Box plots show control data (blue) and experimental data (orange), each dot represents one segment, density of data distribution shown below box plots on y-axis and values along x-axis, control (blue), experimental (red), with significant effects (p < 0.001).

(D-G) Proximal inter-leg distance: Distance between femur-tibia joints of left and right front legs changes during head grooming. P inter-leg distance decreases with 13A activation (D), increases with 13A silencing (E), and decreases with both 13B activation (F) and silencing (G).

(H-K) Frequency modulation: Silencing or activating 13A or 13B neurons reduces median frequency of proximal joints in dusted flies. Increased variability indicated by a broader interquartile interval. 13B silencing reduces median frequency from ∼8 Hz to ∼5 Hz.

13A Neurons Regulate Leg Coordination During Grooming and Walking

Neuronal labeling and connectivity: (A1) A confocal image showing six Dbx positive 13A neurons/hemisegment labeled by GFP driven by R35G04-DBD, Dbx-AD Split GAL4. T1 right front leg neuropil of the VNC is shown. GFP (green) labels 13A neurons. Neuroglian labels axon bundles in magenta. (A2) Schematic illustrating all muscles controlled by the motor neurons inhibited by specific 13A neurons. (A3) Top panel shows confocal images showing multicolor flip out clones (MCFO) (Nern et al., 2015) of 13A neurons. Bottom panel shows corresponding EM reconstructions of 13A neurons that resemble MCFO clones.

Manipulating activity of Six Dbx positive 13A Neurons: Silencing and activation of 13A neurons in dust-covered flies using R35G04-DBD, Dbx-AD Split Gal4 > UAS Kir and UAS CsChrimson, respectively. Control conditions include AD-DBD Empty Split for inactivation and AD-DBD Empty Split with UAS CsChrimson for activation. 13A inactivation (n=12 experimental flies/47 videos), activation (n=19 experimental flies/46 videos). Box plots showing control data in blue and experimental in orange, with the density of the data distribution shown below the box plots on the y-axis and values along the x-axis, control in blue, experimental in red. Significant effects observed in all analyzed cases (p < 0.0001).

(B, C) Reduction in head grooming bout duration (s*10-2) during silencing (B) and activation of 13A neurons (C).

(D,E) Maximum angular velocity (° /s*10-2) of the proximal joint reduces during silencing (D) and and upon continuous activation (E) of 13A neurons.

(F-H’) Joint Positions: Schematic showing position of various joints of the front legs is shown in F. Contour plots (probability distribution of joint positions) of the front legs of all the control and experimental flies during head grooming. The position of leg terminal (tarsus tip) is shown in colors: left leg in blue, right in red, the distal joint in dark and light green, and medial joint in dark blue and maroon. Joints positions are significantly altered (p<0.001) upon activation of 13A neurons (G’), and slightly upon silencing (H’) of 13A neurons in dust-covered flies.

(K) Whole body velocity (pixels[or mm*10-2]/s) decreases upon 13A activation during walking in dust covered flies.

Two Dbx Positive 13A Neurons Are Involved in Leg Coordination During Grooming in Dust-Covered Flies

(A) Confocal image showing two Dbx positive 13A neurons/hemisegment labeled by GFP driven by R11C07-DBD, Dbx-AD Split GAL4 in the adult VNC, labeled by GFP (green). nC82 (magenta) labels synaptic neuropil.

(B-G) Effects of manipulating activity of two 13A neurons: Silencing and activation experiments in dusted flies using R11C07-DBD, Dbx-AD Split Gal4 > UAS GTACR1 and UAS CsChrimson, respectively. Control conditions include AD-DBD Empty Split with UAS GTACR1 for inactivation and with UAS CsChrimson for activation. 13A inactivation (n=7 experimental flies/40 videos), activation (n=4 experimental flies/18 videos). Significant effects observed in all analyzed cases (p < 0.0001).

(B-D’) Contour plots (probability distribution of joint positions) of the front legs during grooming actions. Joints positions are significantly altered upon silencing (C’) and activation of 13A neurons (D’) in dust covered flies. Joint positions are shown during head sweeps (C-D’).

(E,E’) Median frequency (Hz) of the proximal and medial joints decreases upon silencing of two 13A neurons

(F) Maximum angular velocity (° /s*10-2) of the proximal joint reduces upon silencing of two 13A neurons.

Neuronal Labeling. Connectivity and Behavior of Specific 13B Neurons

(A) Neuronal labeling: A confocal image showing clonal analysis of 2-3 13B neurons labeled in each hemisegment of the adult ventral nerve cord by R11B07-DBD, GAD-AD Split GAL4. Multicolor flip-out (MCFO) clones of all the 13B neurons labeled are shown in purple, green and black.

(B) MCFO clones of individual 13B neurons labeled in the right T1.

(B’) EM reconstructions of 13B neurons in right T1 that resemble these MCFO clones.

(C) Muscle targets disinhibited by two of these 13B neurons and those inhibited by one 13B neuron. Proximal extensor MNs and medial flexor MNs are disinhibited while medial extensors are inhibited. Extensor muscles in orange and flexor in blue.

(D-E’) Contour plots (probability distribution of joint positions) of the front legs during grooming actions. Joints positions are significantly altered upon silencing (D’) and activation of 13B neurons (E’) in dust covered flies. Joint positions are shown during head sweeps (D-E’’).

(F-G) Reduction in head grooming bout duration (s*10-2) upon silencing (F) and activation of 13B neurons (G).

Pulsed Activation of 13A Neurons Triggers Rhythmic Actions in Clean Undusted Flies

(A) Schematic showing proximal joint angles of left (PL) and right (PR) legs

(B) Left-right coordination and muscle synergies during anterior grooming. Dusted flies perform alternating leg rubs and head sweeps. Proximal joint angular velocities are shown. PL (blue) and PR (red) joints move anti-phase during leg rubs and in-phase during head sweeps (highlighted yellow box). Positive values indicate extension, and negative indicates flexion. (B’) Each flexion and extension cycle lasts ∼140 ms, with each phase around 70 ms. (B’’) Maximum angular velocity of proximal joint during head sweeps.

(C-F) Effect of optogenetic activation using 70ms on and 70ms off pulses in specific 13A neurons (R35G04-DBD, Dbx-GAL4-AD >UAS CsChrimson) in undusted flies.

(C) Angular velocity of PL and PR leg joints shows anti-phase leg rubs and sustained in-phase head sweeps, with light pulses active from time=0.

(D) Behavioral ethogram showing various grooming actions and walking triggered by 70ms on and 70ms off pulsed activation of 13A neurons in undusted flies, with light pulses on from time=0.

(E) Joint Positions: Contour plots (probability distribution of joint positions) of front legs in undusted flies during head grooming (E) and walking (E’) upon pulsed 13A activation, with tarsus tip positions in blue for left leg (L) and red for right leg (R), distal joints in dark and light green, and the medial joint in dark blue and maroon.

(F) Maximum angular velocity of proximal joint during head sweeps upon pulsed 13A activation in undusted flies, comparable to that observed in dusted flies (B’’).

Activation of six 13A neurons with 70ms on and 70ms off pulses in undusted flies induces grooming and walking behaviors.

In this video, light pulses start at t=30s with a pattern of 70ms on and 70ms off. Light is off from t=0-30s.

Modeling the 13A Circuits.

(A) Circuit diagram showing inhibitory circuits and synaptic weights based on connectome.

(B) Adjacency matrices of the model circuits (after fine-tuning).

(C) Adjacency matrices from the empirically estimated weights, indicated in the simplified circuit diagram in (A).

(D) The three “joint” angles of the left leg (left) and the right leg (right) as they change over the time of one episode (500 frames). Colors indicate “joint” angles in the same order as in (I).

(E) Same as (D) but zoomed-in to between 100-200 frames.

(F) Same as (E) but showing angular velocities.

(G) Firing rates (activity levels) of the two 13A neurons over one episode (500 frames), for both legs (left, right).

(H) Same as (G) but zoomed-in to between 100-200 frames.

(I) Video frames from the beginning, middle, and end of a video of one episode. Left leg is represented by three “joints”: distal (cyan), medial (pink), and proximal (blue). Right leg: distal (purple), medial (orange), and proximal (red). The legs originate from the “base” (yellow). As legs move over the “body” (the environment – dust is represented as the green Gaussian distribution), the dust (green) is getting removed (black background). The bottom of each movie frame shows the activity of the two left 13A nodes and six left MNs (blue). The right leg nodes are shown in red, on the right side. Brightness of the nodes indicates the activity level. See Video1.

(J) The dynamics of angular velocities of the left leg’s “joints”, and left 13A activation levels, over 100 episodes (500 frames each), when no stimulus is given (indicated by empty matrix on the top). Each row of each matrix is one episode.

(K) Same as in J, but stimulation with pulses of varying durations is given. Top row of each matrix: pulse duration=2 frames; bottom (100th) row of each matrix: pulse duration=100 frames. The pulse stimulation is indicated in the top matrix.

Modeling the 13A Circuits.

(A) Dynamics of angular velocities and 13A neurons, when no stimulus is given (Similar to Figure 7 J).

(B) Same as A, but with proprioceptive SN → MN feedback connections obliterated.

Modeling the 13A circuits.

Description in Figure 6I.