henhaobio
Sign in

bioRxivNeurosciencePreprint

A Premotor Excitation-Inhibition Motif Shapes Protopodium Dynamics During Drosophila Larval Forward Crawling

Ankura SitaulaiD, Lizzy OlseniD, Dulanjana M. PereraiD, Yuhan HuangiD, Vashisht Errabolu Meher, Parinita Mitchelle Mandhyan, Nathan ByrdiD, Sara Arredondo, Lauren Carlisle, Yufeng Wan, Isuru S. GodageiD, Aref Arzan ZariniD

PDFDOI
In-depth full-text digest available · Jump to digest ↓

Abstract

Animals use shared muscles and motor neurons to generate distinct movements, but how premotor circuits differentially recruit these neuromuscular components remains unclear. Drosophila larvae crawl forward and backward using overlapping motor pools, yet the timing of ventral oblique (VO) muscle activity differs between these behaviors. Here, we show that VO muscles are major contributors to the movement of protopodia, limb-like structures that help larvae interact with the crawling surface. Muscle imaging, perturbation, and modeling indicate that VO activity is particularly important for protopodium folding, whereas contractions of both VO and ventral longitudinal (VL) muscles contribute to protopodium stride length and crawling efficiency. We identify a tri-segmental premotor motif--comprising excitatory (A27h, A18b3) and inhibitory (A06c) neurons--that shapes VO timing during forward crawling. A06c exhibits two activity peaks during forward fictive locomotion, defining a constrained window of VO MN activation, whereas during backward locomotion the early A06c peak is absent and the excitatory PMNs A27h and A18b3 are inactive. Disrupting this motif shifts VO activity earlier, reduces intersegmental timing delays, increases temporal overlap of VO activity, and impairs forward protopodium folding, stride, and crawling efficiency. These findings show how premotor excitation and inhibition coordinate muscle timing to shape locomotor mechanics.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (22 Aug 2026) · CC BY 4.0

In Drosophila larvae, ventral oblique (VO) muscles drive protopodium folding, and a tri-segmental A27h–A18b3–A06c premotor excitation-inhibition motif restricts their activation to a late, forward-specific window.

Forward and backward larval crawling recruit largely shared motor neurons and muscles, yet ventral oblique (VO) muscles contract late in forward and early in backward crawling; prior work described this timing difference but not the circuit imposing it or its mechanical role. Here VO function is tested with causal manipulations, and a connectome-defined, functionally validated premotor motif is linked to muscle timing, protopodium mechanics and crawling efficiency.

Key results

  1. ShownVO muscles are sufficient and necessary for protopodium folding, with little redundancy: silencing just one of the two tonic VO motor neurons impairs folding and stride.

    Chrimson activation of VO-MN15–17 (NB7-1, N = 14; FD4, N = 9) gave a ventral C-bend with protopodium folding; ventral longitudinal (VL)-only activation (HB9) gave body shortening (N = 26) and the weakest contraction and folding indices (12 trials, 8 animals; Kruskal–Wallis/Dunn). GtACR1 silencing of MN15–17 alone lowered stride and folding index (N = 5 per genotype); tonic VL silencing had minimal effect. · The activity of VO muscles is sufficient to…; VO muscles are necessary for protopodium folding…; Figure legends

  2. ShownDuring forward fictive crawling the GABAergic premotor neuron A06c fires twice, before and after motor neuron activity, with excitatory A27h peaking in between; in backward crawling A27h and A18b3 are silent and A06c's early peak is absent.

    Dual-colour calcium imaging (jRCaMP1b in premotor neurons, GCaMP6m in reference Ib MNs 2, 3, 4, 9, 10) of isolated CNS. Intersegmentally, A27h (n+2) and A18b3 (n+1) fired with the first A06c (n) peak (new A18b3/A27h set: 15 fictive waves, 4 animals); sample sizes for other pairings are not given in the text. · The coordinated activity of A06c, A18b3, and…; Calcium imaging in neurons; Figure legends

  3. ShownSilencing A06c, A27h or A18b3 makes VO activation premature and more synchronous across segments, reduces protopodium folding and stride, and lowers crawling efficiency; silencing two of them gives the strongest defects.

    GtACR1 silencing shortened VO onset and peak delays and raised half-maximum-window and area overlap between adjacent and alternate segments, and moved VO onset toward muscle M26 (N = 5–15 larvae; n = 12–96 and 19–80 bouts). Folding index and stride fell (N = 9–22 larvae, 30–107 protopodia); distance per cycle fell most with dual silencing (n = 40 larvae per genotype; Wilcoxon, Holm). · Disrupting the A27h–A18b3–A06c motif compromises…; Premature VO muscle activation compromises…; Muscle GCaMP Data Analysis; Figure legends

  4. ShownElectron microscopy (EM) reconstruction identifies A06c (inhibitory) and A27h (excitatory) as the main inputs to tonic VO motor neurons and a tri-segmental A27h→A18b3→A06c path; optogenetics under tetrodotoxin confirms these links are monosynaptic.

    Across six hemisegments (A1–A3) of a first-instar EM volume, A06c supplied 17.5%, 18% and 23% of all synapses onto VO MNs and A27h 10%, 10% and 13%; A27h also contacts A06c (incoherent type-1 feedforward loop). Chrimson activation of A18b3 or A27h raised GCaMP6m in A06c or A18b3 with and without 3 µM tetrodotoxin (TTX); controls lacking the driver showed no response. · VO MNs receive excitatory and inhibitory inputs…; The connectome-based monosynaptic connections…; Optogenetic validation of EM connectome; Electron microscopy and CATMAID reconstructions

  5. SuggestedPlanar four-bar kinematic and nine-segment robotic models predict that VO shortening dominates protopodium gait, with VL shortening contributing comparatively little under the modelled constraints.

    Inverse kinematics from tracking of 5 larvae matched measured VO length changes but predicted much smaller VL shortening than observed. Robot advance per cycle: 48.26% of a segment width with in vivo-derived timing, 22.37% with VO silenced, 52.12% with VL silenced (called insignificant), 71.88% with model-predicted dynamics. · Mechanical modeling predicts a dominant role for…; Modeling overview; Figure legends

Methods, data and reproducibility

Motor neurons were activated (Chrimson) or silenced (GtACR1), and premotor neurons silenced, in intact larvae during muscle GCaMP and/or brightfield protopodium imaging; crawling efficiency was scored in agarose grooves. VO premotor inputs were traced in an L1 EM volume (CATMAID) and tested by dual-colour calcium imaging and TTX-controlled optogenetics in isolated CNS, plus four-bar kinematic and robotic models.

  • Intact larvae imaged crawling forward and backward (muscle GCaMP6f plus brightfield protopodia); forward vs backward onset comparison reuses Zarin et al. 2019 data · Drosophila melanogaster · n = 16 forward and 17 backward bouts, 7 animals each (Figure 2)
  • Forward-crawling muscle and protopodium tracking used to drive the four-bar kinematic model · Drosophila melanogaster · n = 5 larvae
  • Optogenetic motor neuron activation (Chrimson): whole-body posture and confocal VO length/protopodium width · Drosophila melanogaster · n = Posture: NB7-1 14, FD4 9, Nkx6 split 23, HB9 26, CQ 11 larvae
  • Motor neuron silencing (GtACR1): crawling efficiency in agarose grooves and protopodium folding/stride · Drosophila melanogaster · n = 10–22 larvae per genotype (efficiency); 5 per genotype (protopodia)
  • TEM volume of a first-instar (L1) larval nervous system (Ohyama et al., 2015); VO motor neuron premotor inputs reconstructed in CATMAID · Drosophila melanogaster · n = six hemisegments (A1–A3)
  • Dual-colour calcium imaging of A06c, A27h and A18b3 against reference motor neurons in isolated CNS (fictive crawling) · Drosophila melanogaster · n = 15 fictive waves, 4 animals (A18b3 vs A27h set); others not stated
  • Premotor neuron silencing with muscle GCaMP6f imaging of intersegmental and intrasegmental VO timing · Drosophila melanogaster · n = N = 5–15 larvae; n = 12–96 and 19–80 bouts
  • Premotor neuron silencing: protopodium folding/stride and crawling efficiency · Drosophila melanogaster · n = 9–22 larvae, 30–107 protopodia; 40 larvae per genotype (efficiency)
Methods & tools
GCaMP6f, GCaMP6m, jRCaMP1b, Chrimson, GtACR1, Gal4, split-Gal4 and LexA drivers, MultiColor FlpOut (MCFO), CATMAID, Zeiss LSM900 confocal microscope, MATLAB (custom scripts), Fiji, G*Power 3.1
Reproducibility gaps
Raw imaging and behaviour data and custom MATLAB/R code are promised for Zenodo, but no link is given. The L1 EM volume is available on request from Albert Cardona. Fly stocks and antibodies are listed in a Key Resources Table that is not part of the text.
Limitations (6)
  • The kinematic and robotic models address only protopodium mechanics: they do not cover VL roles in visceral pistoning and intersegmental body mass transport, or whole-body force transmission and internal mass redistribution. · stated by the authors
  • A27h and A18b3 also feed other premotor pathways (e.g., A27j2/Gdl, A14a), so the stronger defects after dual silencing likely include effects beyond VO timing. · stated by the authors
  • 36G02-Gal4 also labels M and A03g neurons, and 47E12-Gal4 labels A18a and occasional sensory neurons; for the protopodium assays an A27h split-Gal4 line was additionally used, and phenotypes were confirmed with an A18b3 split-Gal4 line. · stated by the authors
  • Kinematic fits show minor offsets attributed to unmodeled tissue deformation, and robot runs with model-predicted dynamics gave 71.88% displacement, attributed to mismatch between modelled elastic elements and real tissue. · stated by the authors
  • Premotor wiring comes from one first-instar EM volume (six hemisegments, A1–A3) and premotor activity from fictive crawling in isolated CNS, whereas the muscle and behaviour experiments used L2–L3 larvae. · noted in this summary
  • In the muscle-timing experiments, A27h was silenced only with 36G02-Gal4, which also labels M and A03g neurons, so off-target silencing could contribute to the VO timing changes attributed to A27h. · noted in this summary

Read this if you study how premotor excitation and inhibition set muscle timing across behaviours, or want a Drosophila larval example tying EM connectomics to calcium imaging, optogenetics and biomechanical modelling.

Citations
2

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISHuang '22Zarin '19He '26Cooney '23Greaney '26Yoon '19
85% semantic matchNeuroscience
Yuhan Huang, Aref Arzan Zarin·bioRxiv·Aug 18, 2022· 7 citations