Inhibitory circuits control leg movements during Drosophila grooming
- Neuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, United States
Figures
Figure 1 with 2 supplements
Anatomical Distribution and Behavioral Contributions of 13 A and 13B Hemilineages.
Schematic showing segmental distribution of 13 A (green) and 13B (cyan) neurons across pro-, meta-, and meso-thoracic segments (T1, T2, T3) of VNC. Confocal image: Six GABAergic 13 A 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. EM reconstructions: 62 13 A neurons (green) and 64 13B neurons (cyan) in right T1. Ventral side up. (A) Continuous activation of 13 A and 13B neurons labeled by R35G04-GAL4-DBD, GAD-GAL4-AD in dusted flies reduces front leg rubbing and head sweeps and induces unusual leg extensions. Control: AD-DBD EMPTY SPLIT >UAS CsChrimson (gray). Experiment: R35G04-GAL4-DBD, GAD-GAL4-AD>UAS CsChrimson (red). Box plots indicate the percentage of time dusted fly engaged in a given behavior over a 4-min assay (n=7). The solid blue line marks the mean, dark shading the 95% confidence interval, red dashed line the median, and light shading ± 1 standard deviation. ***p≤0.001, *p≤0.05. (E-F) Continuous activation of 13 A and 13B neuron subsets induces front leg extension in headless flies. (E, E′) Representative video frames showing headless flies (dusted and undusted) with extended front legs (orange arrowhead) following continuous optogenetic activation of neurons labeled with R35G04-GAL4-DBD, GAD-GAL4-AD>UAS- CsChrimson. Dashed box in E highlights the front legs; schematic illustrates the extended posture. (F) Quantification of leg extension phenotypes in dusted and undusted headless flies. Bar plots show the percentage of flies displaying leg extension (red) or a normal posture (gray). Percentages are calculated as the number of flies showing each posture divided by the total number of flies per condition. Dusted: n=9; undusted: n = 5. (G–H) Silencing 13 A and 13B neuron subsets locks front legs in flexion in headless flies. (G, G′) Representative video frames showing dusted and undusted headless flies with sustained front leg flexion following silencing of neurons labeled with R35G04-GAL4-DBD, GAD-GAL4-AD>UAS TNTe. Blue arrowheads indicate the flexed posture. (H) Quantification of leg flexion phenotypes in dusted and undusted headless flies. Bar plots show the percentage of flies displaying sustained flexion (red). All flies (100%) in both dusted (n=13) and undusted (n=9) conditions showed the phenotype. Also see Figure 1—video 1.
Figure 1—figure supplement 1
Expression pattern in the central nervous system of various lines used for behavior experiments.
R35G04-DBD and GAD1-AD Split GAL4 combination labels approximately 6–7 13 A 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 nine neurons per hemisegment (possibly 13 A neurons with posterior cell bodies Soffers et al., 2025; Marin et al., 2024,or 3B neurons) and four neurons per hemisegment (possibly 0 A) within the Accessory Mesothoracic neuropil (AMNp) and T2 midline. R35G04-DBD and Dbx-AD Split GAL4 combination specifically labels a subset of 13 A neurons already included in the R35G04-DBD and GAD1-AD Split GAL4 line, thereby isolating 13 A neurons without labeling 13B neurons. It also labels approximately three neurons per hemisegment with posterior cell bodies. (A) R11B07-DBD and GAD1-AD Split GAL4 combination labels three 13B neurons. It occasionally labels two ectopic ascending neurons per hemisegment.
Figure 1—video 1
Manipulating the activity of six 13 A neurons and six 13 A neurons in headless flies.
Inactivation: Legs locked in flexion in clean and dust-covered flies. Activation: legs extended in clean and dust-covered flies.
Figure 2 with 7 supplements
Spatial map of premotor 13 A neurons correlates with their connections to motor neurons (MNs).
Hierarchical clustering of 13 A hemilineage. Clustering of 13 A neuron types in the right T1 segment was performed using NBLAST, resulting in identification of 10 morphological groups or clusters. EM reconstructions of distinct 13 A clusters are shown. Neurons are named based on morphological clustering. For example, all neurons in the 13 A-3 cluster have similar morphology, with 10 neurons labeled as 13 A-3 (a–j) (olive). Images of each 13 A neuron are shown in Figure 2—figure supplement 1. Also see Figure 2—video 2. A=anterior, L=Lateral. Ventral side is up. (A) Cosine similarity graph showing pairwise similarity between 13 A neurons based on their MN connectivity patterns. 13 A neurons are organized based on anatomical clusters obtained with NBLAST as described above. It depicts a correlation between the anatomy of 13 A neurons and their connections to MNs. For example, 13 A-1a, –1b, –1 c, –1d (cluster 1) connect to the same set of MNs, therefore have high cosine similarity with each other (as seen across the diagonal). The graph also gives insights into groups of 13As that control similar muscles. For example, cluster 1 neurons have high cosine similarity with cluster 3 13 A neurons (while, 3 g neuron is an exception).
Figure 2—figure supplement 1
Spatial map and connectivity of premotor 13 A neurons.
(A1–A6) Top: EM reconstruction showing morphology of individual 13 A 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 13 A neurons. (A7) Top: EM reconstruction showing morphology of all cluster-1 13 A neurons combined. Bottom: Connectivity matrix showing cluster 1 13 A neurons and their MN connections. These neurons connect to sternotrochanter extensor (SE) and trochanter extensor (Tr E) motor neurons. Edges represent synaptic weight. (B1–B6) Top: EM reconstruction showing morphology of individual Cluster-3 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-3 13 A neurons. (B7) Top: Morphology of Cluster-3 13 A neurons combined except 13 A-R-3g. Bottom: Connectivity matrix showing connections between cluster-3 13 A neurons and MNs. Tr. = trochanter, Tr E=trochanter extensor, Tr F=trochanter flexor, Ta D=tarsus depressor. (B8) Top: Morphology of Cluster-2 13 A neuron. Bottom: Leg schematic for muscles innervated by the Cluster-2 13 A neuron. (C1–C5) Top: EM reconstruction showing morphology of individual Cluster-4 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-4 13 A neurons. (C6) Top: Morphology of Cluster-4 13 A neurons combined. Bottom: Connectivity matrix showing connections between cluster-4 13 A neurons and MNs. (D1–D8) Top: EM reconstruction showing morphology of individual Cluster-5 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-5 13 A neurons. (D9) Top: Morphology of Cluster-5 13 A neurons combined. Bottom: Connectivity matrix showing connections between cluster-5 13 A neurons and MNs. Fe R=femur reductor. (E1–E6) Top: EM reconstruction showing morphology of individual Cluster-6 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-6 13 A neurons. (E7) Left: Morphology of Cluster-6 13 A neurons combined. Right: Connectivity matrix showing connections between cluster-6 13 A neurons and MNs. (F1–F3) Top: EM reconstruction showing morphology of individual Cluster-7 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-7 13 A neurons. (F4) Top: Morphology of Cluster-7 13 A neurons combined. Bottom: Connectivity matrix showing connections between cluster-7 13 A neurons and MNs. (G1–G5) Top: EM reconstruction showing morphology of individual Cluster-8 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-8 13 A neurons. (G6) Top: Morphology of Cluster-7 13 A neurons combined. Bottom: Connectivity matrix showing connections between cluster-7 13 A neurons and MNs. (H1–H8) Top: EM reconstruction showing morphology of individual Cluster-9 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-9 13 A neurons. (H9) Top: Morphology of Cluster-9 13 A neurons combined. Bottom: Connectivity matrix showing connections between cluster-9 13 A neurons and MNs. (I1–I8) Top: EM reconstruction showing morphology of individual Cluster-10 13 A neurons. Bottom: Leg schematic highlighting muscles innervated by the MNs inhibited by Cluster-10 13 A neurons. (I9) Top: Morphology of Cluster-10 13 A neurons combined. Bottom: Connectivity matrix showing connections between cluster-10 13 A neurons and MNs. Protractors/levators/extensors in orange and retractors, depressors, flexors in blue.
Figure 2—figure supplement 2
Connectivity matrix of 13 A neurons and the motor neurons showing left-right comparison and spatial map.
The 13 A neurons that belong to the same anatomical cluster connect to the same set of motor neurons (MNs). 13 A 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. Clusters are matched as groups, but individual neurons within a cluster are not necessarily one-to-one homologs across sides. The edge width between 13 A 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 left (Soler et al., 2004) 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. Protractors/levators/extensors: orange, retractors, depressors, flexors: blue.
Figure 2—figure supplement 3
Anatomical classification of 13B neurons.
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. For example, all neurons in the 13B-1 series have similar morphology and contain eight neurons 13B-1(a-h). The right panel shows morphologies of neurons in each cluster in T1. Cell bodies are towards the left side of the midline, but all axonal and dendritic projections are towards the right T1. Each subplot depicts a distinct cluster, with different colors indicating different clusters. A=anterior, P=posterior, L=left, R=right. Ventral side is up. (A) 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.
Figure 2—figure supplement 4
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 21 A, 24B (yellow), cholinergic hemilineages 7B, 3 A, 20 A (green), GABAergic hemilineages 12B (gray), and other unknown neurons (blue, orange).
Figure 2—video 1
13 A neurons in the right front leg neuromere (T1R).
Primary neurons in brown. Secondary neurons in green and red. Three sub-bundles of hemilineage 13 A neurons are shown across 2D EM sections (left) and 3D rendering (right) of the cell bodies and 13 A axonal tracks entering the neuropil.
Figure 2—video 2
13 A morphological clusters.
Figure 2—video 3
13 A cluster 6 neurons (red) and downstream Tibia extensor MNs (feti and seti, green).
Figure 3 with 3 supplements
Inhibitory circuitry for antagonistic muscle control.
Schematic of inhibitory circuit motifs. (A1) Feedforward inhibition by 13 A/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 1296 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 13 A groups that inhibit antagonistic MNs may induce flexor-extensor alternation. 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 13 A neurons (13As-i) in blue, and extensor-inhibiting 13As (13As-ii) in orange. The thickness of edges between nodes is determined by the number of synapses. Node colors were assigned based on the type of neurons, with specific colors denoting different subtypes of 13 A/B neurons and MNs. Feedforward inhibition: Primary neurons (13A-10f-α, 9d-γ, and 10e-δ) and 13 A-10c (13As-i) connect to tibia flexor MNs (blue edges), making a total of 85, 219, 155, and 157 synapses, respectively. Twelve secondary 13As ii inhibit tibia extensor MNs (orange edges), with strong connections from 13 A-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 a combination of proximal (sternotrochanter, tergotrochanter, trochanter extensor, tergoplural promotor), medial (tibia flexor), and distal (tarsus depressor) MNs, while secondary 13As (9e and 9f) target antagonist MNs including sternal posterior rotator, pleural remotor abductor, and tibia extensor. Reciprocal connections between them indicate that generalist 13As coordinate multi-joint muscle synergies through inhibition of antagonistic motor groups. Leg schematic shows the muscles innervated by the corresponding MNs in various leg segments (Th = thorax, C = coxa, Tr = trochanter, Fe = femur, Ti = tibia, Ta = tarsus). (Data for 13A-MN connections are shown in Figure 2—figure supplement 1I9, I6, I7, H9, H4, and H5; 13A-13A connections shown in Figure 3—figure supplement 1C). 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. Extension-sensing proprioceptors target tibia flexor MNs and two 13As (13As-ii) that inhibit extensor MNs. Claw extension neurons also connect to 13A-δ. One 13B that disinhibits flexor MNs also receives connection from extension-sensing proprioceptors. Also see Figure 3—figure supplement 3.
Figure 3—figure supplement 1
Disinhibition matrix.
Network visualization of all 13 A and 13B neuronal interconnections. Network graph showing all 13B (presynaptic) to 13 A (postsynaptic) connections (blue edges), 13A to 13A connections (red edges), 13 A to 13B connections (gray edges) and 13B to 13B connections (green edges). 13 A nodes in red. 13B nodes in blue. Disinhibition mediated by 13B neurons: A connectivity graph showing all 13B to 13 A connections. The leg schematic on the right side shows targets of motor neurons inhibited by these 13 A 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 13 A targets. Protractors/levators/extensors in orange and retractors, depressors, flexors in blue. Disinhibition mediated by 13 A-13A connections: A connectivity graph highlighting connections within 13 A neurons that leads to disinhibition of motor neurons. Nodes of the same color represent the same 13 A anatomical cluster. Primary 13 A 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. Disinhibition mediated by 13A-13B connections: A connectivity graph showing connections from 13 A neurons to 13B neurons. Note that most of these postsynaptic 13B neurons are premotor (shown in panel F). Disinhibition mediated by 13B-13B connections: A connectivity graph showing interconnections between 13B neurons. 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. Protractors/levators/extensors in orange and retractors, depressors, flexors in blue.
Figure 3—figure supplement 2
Neurons Downstream of a 13B Neuron (13B-4i).
13 A neurons (red) comprise 36.8% of the total downstream synapses. 20/22 A cholinergic neurons (green), 24.5% of the total synapses. 9 A neurons (cyan)(6.5%), two contralateral interneurons (9%), 13B neuron (1.9%), 3B (1.9%), ipsilateral interneurons (possibly 21 A?) (11%) of the total downstream partners. (A) Individual downstream neurons. (B) Aggregate of the same type of neurons.
Figure 3—figure supplement 3
Sensory feedback onto inhibitory 13 A neurons and motor neurons.
Connectivity matrix showing position sensing claw neurons and motion sensing hook neurons send feedback connections to 13 A neurons and antagonistic MNs. Flexion position and motion sensing proprioceptors (blue) neurons connect to tibia extensor MNs (orange) and primary 13 A neurons (13As-i group) (blue). 13As inhibit tibia flexor MNs. Thus, when the flexion is complete, it could induce extension and inhibit flexion via 13 A neurons. Similarly, extension position and motion sensing proprioceptors (orange) neurons connect to tibia flexor MNs (blue) and two 13 A neurons (13As-ii group) (orange) 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. Edge color corresponds to the identity of the proprioceptive neuron (schematic in Figure 3D).
Figure 4 with 3 supplements
13 A and 13B neurons are required for leg coordination during grooming.
(A-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. The schematic (right) indicates the corresponding joint angles. (A’-A”) The proximal and medial joint movements within a leg occur effectively in phase, with a mean lag of ~0.8 frames (8ms) during leg rubbing (A′) and during head grooming sweeps (A″). Bar plots show the lag; each dot indicates one animal. Frame = 10ms. Neuronal labeling of 13A and 13B neurons. Top: Confocal image of six Dbx positive 13 A neurons per hemisegment labeled by GFP using R35G04-GAL4-DBD, Dbx-GAL4-AD in VNC. Neuroglian (magenta) labels axon bundles. Bottom: Confocal image of three 13B neurons per hemisegment labeled by GFP using R11B07-GAL4-DBD, GAD-GAL4-AD. Nc82 (magenta) labels neuropil. (C–I) Effects of neuronal activity manipulation in dusted flies. Silencing and activation of 13 A neurons in dusted flies using R35G04-GAL4-DBD, Dbx-GAL4-AD with UAS Kir or UAS CsChrimson, respectively (n=12 silencing, n=19 activation). Control: AD-GAL4-DBD EMPTY SPLIT with UAS Kir or UAS CsChrimson. For 13B neurons, R11B07-GAL4-DBD, GAD-GAL4-AD with UAS GtACR1, or UAS CsChrimson, respectively (n=7 silencing, n=9 activation); control: AD-GAL4-DBD EMPTY SPLIT with UAS GtACR1 or UAS CsChrimson. Each panel compares control (blue) and experimental (orange) groups. Each dot represents the mean feature value for a single fly. Bars indicate the group mean, and whiskers represent the 95% confidence interval of the group mean. P-values (raw and false discovery rate [FDR]–corrected) are shown above each panel. (C–D) Proximal inter-leg distance: Silencing of 13 A (C) or 13B (D) neurons during head grooming reduces the distance between the femur-tibia joints of the left and right front legs. (E–I) Frequency modulation: Silencing 13 A or 13B neurons reduces mean frequency of proximal joint oscillations in dusted flies. (F, G). Activation of 13 A neurons reduced frequency, although this change did not survive FDR correction. However, continuous activation of 13 A and 13B neurons increased variability in frequency. (H, I). Mean of the per-animal standard deviation (STD) that reflects variability or spread of data is shown.
Figure 4—figure supplement 1
13 A neurons regulate leg coordination during grooming.
Neuronal labeling and connectivity: (A1) A confocal image showing six Dbx positive 13 A 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 13 A neurons. Neuroglial labels axon bundles in magenta. (A2) Schematic illustrating all muscles controlled by the motor neurons inhibited by specific 13 A neurons. (A3) Top panel shows confocal images showing multicolor flip-out clones (MCFO) of 13 A neurons. The bottom panel shows corresponding EM reconstructions of 13 A neurons that resemble MCFO clones. We found four morphologically distinct targeted neurons among six 13 A neurons. This may reflect incomplete labeling or morphological similarity between two neurons that cannot be reliably distinguished. In the latter case, the two neurons likely belong to the same anatomical cluster, hence connect to the same set of MNs. Manipulating activity of six Dbx-positive 13 A neurons: Silencing and activation of 13 A 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. 13 A inactivation (n=13 flies), activation (n=19 flies). Each panel compares control (blue) and experimental (orange) groups. Each dot represents the mean feature value for a single fly. Bars indicate the group mean, and whiskers represent the 95% confidence interval of the group mean. p-values (raw and FDR–corrected) are shown above each panel. (B, C) Reduction in head grooming bout duration (s*10[-2]) and maximum angular velocity (° /s*10[-2]) of the proximal joint upon silencing of 13 A neurons. (D-F’) 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 the 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. Joint positions are significantly altered upon activation of 13 A neurons (E’), and slightly upon silencing (F’) of 13 A neurons in dust-covered flies. (A) Walking in dust-covered flies: Silencing 13 A neurons did not affect the frequency of proximal joint movements (top panel), but reduced the distance between the tarsal tips of the front legs (bottom panel).
Figure 4—figure supplement 2
Two Dbx-positive 13 A neurons are involved in leg coordination during grooming in dust-covered flies.
Confocal image showing two Dbx-positive 13 A 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 13 A 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. 13 A inactivation (n=11), activation (n=4). Bar plots in each panel compare control (blue) and experimental (orange) groups. Each dot represents the mean feature value for a single fly. Bars indicate the group mean, and whiskers represent the 95% confidence interval of the group mean. p-Values (raw and FDR–corrected) are shown above each panel. (C-D’) Contour plots (probability distribution of joint positions) of the front legs during grooming actions. Joint positions are significantly altered upon silencing (C’) and activation of 13 A 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 13 A neurons. (F) Maximum angular velocity (° /s*10[-2]) of the proximal joint does not significantly reduce upon silencing of two 13 A neurons.
Figure 4—figure supplement 3
Neuronal labeling.
Connectivity and behavior of specific 13B neurons. 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. 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. (A) Muscle targets disinhibited by two of these 13B neurons and those inhibited by one 13B neuron. Proximal (Th-C, C–Tr) extensor MNs and medial (Fe-Ti) flexor MNs are disinhibited while medial extensors are inhibited. Protractors/levators/extensors in orange and retractors, depressors, flexors in blue. (D-E’) Contour plots (probability distribution of joint positions) of the front legs during grooming actions. Joint 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, F’) Mean duration of head grooming bouts is longer upon silencing 13B neurons, whereas continuous activation of 13B neurons causes a slight, non-significant decrease in leg rubbing bouts in dusted flies. Bar plots compare control (blue) and experimental (orange) groups. Each dot represents the mean feature value for a single fly. Bars indicate the group mean, and whiskers represent the 95% confidence interval of the group mean. p-Values (raw and FDR–corrected) are shown above each panel. (G,G’) Percentage of time spent doing anterior grooming is reduced both upon continuous silencing and activation of 13B neurons. Box plots indicate the percentage of time dusted flies engaged in a given behavior. The solid blue line marks the mean, dark shading the 95% confidence interval, red dashed line the median, and light shading ± 1 standard deviation. ***p≤0.001, **p≤0.01. Silencing experiment total time = 18 min, scored with ABRS. Activation experiment total time = 4 min, manually scored due to erratic leg extensions.
Figure 5 with 2 supplements
Pulsed activation of 13 A neurons triggers rhythmic actions in clean undusted flies.
Schematic showing proximal joint angles of left (PL) and right (PR) legs. 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’’) Mean lags between proximal joints of the left and the right legs during leg rubbing and head sweeps. High lag during leg rubbing (left panel) indicates out-of-phase movement, and low lag during head sweeps (right) indicates in-phase movement. Bar plots show the lag; each dot indicates one animal. Frame = 10 ms. (C–F) Effect of optogenetic activation using 70 ms on and 70 ms off pulses in specific 13 A neurons (R35G04- DBD, Dbx-GAL4-AD>UAS CsChrimson) in undusted flies. 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. Behavioral ethogram showing various grooming actions (head, front leg, abdomen, back leg, wing, thorax) and walking triggered by 70 ms on and 70 ms off pulsed activation of 13 A neurons in undusted flies, with light pulses on from time = 0. Maximum angular velocity of proximal joints during head sweeps upon pulsed 13 A activation in undusted flies is comparable to that observed in dusted flies. (A) The frequency of proximal joint movements during leg rubbing (left) and head sweeps (right) induced by 13 A pulsed activation is also similar between dusted and undusted flies. Control flies: AD-DBD EMPTY SPLIT >UAS-CsChrimson, dusted; Experimental flies: R35G04-DBD, Dbx-GAL4-AD>UAS-CsChrimson, undusted. Light pulses were delivered at 70 ms on / 70 ms off. Each panel compares control (blue) and experimental (orange) groups. Each dot represents the mean feature value for a single fly. Bars indicate the group mean, and whiskers represent the 95% confidence interval of the group mean. p-Values (raw and false discovery rate [FDR]–corrected) are shown above each panel.
Figure 5—figure supplement 1
Effect of varying optogenetic stimulation period on proximal joint cycle frequency.
(A) Comparison of mean proximal joint cycle frequency during 10 ms on/off stimulation (50 Hz) versus 70 ms on/off (~7 Hz). Comparison for 50 ms on/off (10 Hz) versus 70 ms on/off. Comparison for 110 ms on/off (~4.5 Hz) versus 70 ms on/off. (A) Comparison for 120 ms on/off (~4 Hz) versus 70 ms on/off. No significant differences were detected in any comparison (linear mixed model, p_FDR >0.05). Bar plots in each panel compare control (~7 Hz, blue) and experimental (orange) groups. Each dot represents the mean value for a single fly. Bars show group means, and whiskers represent the 95% confidence interval of the group mean. p-Values (raw and FDR–corrected) are shown above each panel. These results indicate that pulsed activation triggers the circuit’s intrinsic rhythm rather than pacing it at the stimulation frequency.
Figure 5—video 1
Activation of six 13 A neurons with 70 ms on and 70 ms off pulses in undusted flies induces grooming and walking behaviors.
In this video, light pulses start at t=30 s with a pattern of 70 ms on and 70 ms off. Light is off from t=0–30 s.
Figure 6 with 2 supplements
Modeling the 13 A circuits.
Circuit diagram showing inhibitory circuits and synaptic weights based on connectome. Adjacency matrices from the empirically estimated weights, indicated in the simplified circuit diagram in (A). The 13B neurons in this model do not connect to each other, receive excitatory input from the black box, and only project to the 13As (inhibitory). Their weight matrix, with only two values, is not shown. Excitatory and inhibitory connections are shown in red and blue, respectively. Adjacency matrices of the model circuits are the same as in (B) but after fine-tuning. 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 ‘joints’ as follows: distal (cyan), medial (pink), and proximal (blue) for the left leg. Right leg: distal (purple), medial (orange), and proximal (red). 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). Same as (D) but zoomed-in to between 300 and 400 frames. Same as (E) but showing angular velocities [°/frame]. Firing rates (activity levels) of the two 13 A neurons (red and blue) over one episode (500 frames), for both legs (left, right). Same as (G) but zoomed-in to between 300 and 400 frames. Video frames from the beginning, middle, and end of a video of one episode. The 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 13 A 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 Figure 6—video 1. The dynamics of angular velocities of the left leg’s ‘joints’, and left 13 A 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. (The simulation started running for 50 frames before Time = 0 but it starts with very high peaks which were not plotted here for better visualization.). (A) 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.
Figure 6—figure supplement 1
Modeling the 13 A circuits.
Dynamics of angular velocities and 13 A neurons, when no stimulus is given (Similar to Figure 7J). (The simulation started running for 50 frames before Time = 0 but it starts with very high peaks which were not plotted here for better visualization.). (A) Same as A, but with proprioceptive SN → MN feedback connections obliterated.
Figure 6—video 1
Modeling the 13 A circuits.
Description in Figure 6I.
Figure 7
The modeled 13 A circuits can produce rhythmic behavior and activity without rhythmic external input.
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 ‘joints’ as follows: distal (cyan), medial (pink), and proximal (blue). Right leg: distal (purple), medial (orange), and proximal (red). Same as (A) but showing angular velocities [°/frame]. Firing rates (activity levels) of the two 13 A neurons (red and blue) over one episode (500 frames), for both legs (left, right). (A) Same as (C) but zoomed-in to between 300 and 400 frames.
Figure 8
Summary of inhibitory neuron contributions to leg movement coordination.
Inhibitory neurons from the 13A and 13B hemilineages in the Ventral Nerve Cord connect to combinations of leg motor neurons, coordinating groups of muscles to work together in synergy. Circuit motifs connecting inhibitory neurons may support muscle antagonisms and extensor/flexor alternation.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Chicken polyclonal anti-GFP | Abcam | RRID:AB_300798 | (1:1000) |
| Antibody | Rabbit (Rb) polyclonal anti- GFP | Invitrogen | Cat #A11122; RRID:AB_221569 | (1:1000) |
| Antibody | Mouse (ms) monoclonal anti-Bruchpilot | DSHB | RRID:AB_2314866 | (1:200) |
| Antibody | ms anti-Neuroglian (BP104) | DSHB | RRID:AB_528402 | (1:40) |
| Antibody | Mouse polyclonal anti-V5:DyLight 550 | AbD Serotec | RRID:AB_2687576 | (1:300) |
| Antibody | Rabbit polyclonal anti-HA | Cell Signaling Technologies | RRID:AB_1549585 | (1:300) |
| Antibody | Rat monoclonal anti-FLAG | Novus Bio | RRID:AB_1625982 | (1:200) |
| Antibody | Goat anti-Chicken Alexa Fluor 488 | Invitrogen | RRID:AB_142924 | (1:400) |
| Antibody | Goat anti-rabbit Alexa Fluor 488 | Invitrogen | RRID:AB_143165 | (1:400) |
| Antibody | Goat anti-mouse Alexa Fluor 568 | Invitrogen | RRID:AB_2534072 | (1:400) |
| Antibody | Goat anti-mouse Alexa Fluor 633 | Invitrogen | RRID:AB_2535719 | (1:400) |
| Antibody | Goat anti-rabbit Alexa Fluor 568 | Invitrogen | RRID:AB_143157 | (1:400) |
| Antibody | Goat anti-rat Alexa Fluor 488 | Invitrogen | RRID:AB_2534074 | (1:400) |
| Antibody | Donkey anti-rat Alexa 647 | Jackson ImmunoResearch | RRID:AB_2340694 | (1:400) |
| Chemical compound | Insect-a-slip | Bio Quip Products | Cat#2871 A | |
| Chemical compound | Reactive Yellow 86 | Organic Dyestuffs Corporation | CAS 61951-86-8 | |
| Genetic reagent (Drosophila melanogaster) | R35G04-GAL4-DBD | Bloomington Stock Center (BDSC) | RRID:BDSC_70351 | |
| Genetic reagent (Drosophila melanogaster) | GAD-GAL4-AD | Diao et al., 2015 | ||
| Genetic reagent (Drosophila melanogaster) | GAD-GAL4-DBD | Gift from Haluk Lacin and James Truman | ||
| Genetic reagent (Drosophila melanogaster) | Dbx-GAL4-AD | Gift from Haluk Lacin and James Truman | ||
| Genetic reagent (Drosophila melanogaster) | Dbx-GAL4-DBD | Gift from Haluk Lacin and James Truman | ||
| Genetic reagent (Drosophila melanogaster) | R11C07 AD | BDSC | RRID:BDSC_70533 | |
| Genetic reagent (Drosophila melanogaster) | w[1118]; P{y[+t7.7] w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP40 | BDSC | RRID:BDSC_55135 | |
| Genetic reagent (Drosophila melanogaster) | P{JFRC7-20XUAS-IVS-mCD8::GFP}attp40 | BDSC | RRID:BDSC_32194 | |
| Genetic reagent (Drosophila melanogaster) | UAS-GTACR1 | Gift from Adam Claridge-Chang | ||
| Genetic reagent (Drosophila melanogaster) | w[*]; P{y[+t7.7] w[+mC]=UAS-GtACR1.d.EYFP}attP2 | BDSC | RRID:BDSC_92983 | |
| Genetic reagent (Drosophila melanogaster) | “w[1118] P{y[+t7.7] w[+mC]=R57C10-FLPL}su(Hw)attP8; PBac{y[+mDint2] w[+mC]=10xUAS(FRT.stop)myr::smGdP-HA}VK00005 P{y[+t7.7] w[+mC]=10xUAS(FRT.stop)myr::smGdP-V5-THS –10xUAS(FRT.stop)myr::smGdP-FLAG}su(Hw)attP1"(MCFO3)” | BDSC | RRID:BDSC_64087 | |
| Genetic reagent (Drosophila melanogaster) | 10XUAS-IVS-eGFP-Kir2.1 | von Reyn et al., 2017 | ||
| Genetic reagent (Drosophila melanogaster) | “Control-GAL4-AD-GAL4-DBD EMPTY SPLIT: BPp65ADZp(attp40); BPZpGDBD(attp2)” | BDSC | RRID:BDSC_79603 | |
| Software, algorithm | DeepLabCut | Mathis et al., 2018 | RRID:SCR_021391 | |
| Software, algorithm | Python | RRID:SCR_008394 | ||
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | |
| Software, algorithm | FIJI | Schindelin et al., 2012 | RRID:SCR_002285 | |
| Software, algorithm | Adobe illustrator | RRID:SCR_010279 | ||
| Software, algorithm | Adobe Photoshop | RRID:SCR_014199 | ||
| Software, algorithm | Braincircuits | https://braincircuits.io/app?p=fruitfly_fanc_public | ||
| Software, algorithm | Neuroglancer | Maitin-Shepard et al., 2021 | RRID:SCR_015631 | |
| Software, algorithm | fancr | Azevedo et al., 2024; Jefferis, 2024 | https://github.com/flyconnectome/fancr | |
| Software, algorithm | neuPrint | Plaza et al., 2022 | https://neuprint.janelia.org/ | |
| Software, algorithm | CATMAID | Saalfeld et al., 2009 | RRID:SCR_006278 | |
| Software, algorithm | RStudio | RRID:SCR_000432 |
Additional files
-
Supplementary file 1
13 A and 13B Neurons in the Front Leg Neuromere.
The FANC identification numbers and coordinates, with the best matches in MANC and associated annotations, are listed for left and right neuromeres in the T1 segment.
- https://cdn.elifesciences.org/articles/106446/elife-106446-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/106446/elife-106446-mdarchecklist1-v1.pdf
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Inhibitory circuits control leg movements during Drosophila grooming
eLife 14:RP106446.
https://doi.org/10.7554/eLife.106446.4
