Reconstruction of motor control circuits in adult drosophila using automated transmission electron microscopy
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Abstract
To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.
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Intrinsic neurons of the brain make up three-quarters of the adult fly nervous system.
“Intrinsic neurons of the brain make up three-quarters of the adult fly nervous system54–56 and amount to 85% of brain neurons.”
From Neuronal wiring diagram of an adult brain (2024) · cites this paper as [54]
The Discussion section, “A synapse-resolution adult Drosophila VNC dataset” (page unavailable), says the VNC contains about a third of the neurons in the adult central nervous system. That leaves about two-thirds for the brain, including its intrinsic neurons; the paper does not support their making up three-quarters of the larger, whole nervous system. (LLM, conf=0.82)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredThe adult fruit fly has only ∼ 200,000 neurons in its brain, compared with ∼ 70 million in the mouse brain and ∼ 80 billion in the human brain.
“It has only ∼ 200,000 neurons in its brain 24 (compared with ∼ 70 million in the mouse brain 25 and ∼ 80 billion in the human brain 26 ).”
From NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila (2024) · cites this paper as [25]
The Introduction describes the Drosophila nervous system as small, and the Discussion ('A synapse-resolution adult Drosophila VNC dataset') says the VNC contains about a third of the neurons in the adult central nervous system. Neither gives a count of neurons in the adult fly brain; searches for '200,000' and brain-neuron counts found no supporting passage. The mouse and human figures have separate citations in the citing sentence and are not judged here. (LLM, conf=0.76)
Experimental — accuracy not yet measuredLeg muscles are estimated to be innervated by around 70 MNs in each leg, that originate from ~15 hemilineages.
“These leg muscles are estimated to be innervated by around 70 MNs in each leg, that originate from ~15 hemilineages (Baek and Mann, 2009; Brierley et al., 2012; Phelps et al., 2021).”
From Organization of circuits linking descending input to motor output in the Drosophila Male Adult Nerve Cord connectome (2026) · cites this paper as Phelps et al., 2021
The FANC is the first complete EM volume of the adult Drosophila VNC, providing valuable insight into MN morphology, count and organization in the VNC thoracic segments.
“Of note, the FANC (Phelps et al., 2021) is the first complete EM volume of the adult Drosophila VNC, providing valuable insight into MN morphology, count and organization in the VNC thoracic segments, as well as allowing labs to further reconstruct and investigate VNC circuits of interest.”
From Organization of circuits linking descending input to motor output in the Drosophila Male Adult Nerve Cord connectome (2026) · cites this paper as Phelps et al., 2021
These numbers were similar to those from the FANC dataset, where MNs traced (excluding AbN2-4 and AbNT) numbered 507; we found 510 for the MNs in the same nerves.
“These numbers were similar to those from the FANC dataset, where MNs traced (which excluded AbN2-4 and AbNT MNs) numbered 507 (Phelps et al., 2021)—we found 510 for the MNs in the same nerves.”
From Organization of circuits linking descending input to motor output in the Drosophila Male Adult Nerve Cord connectome (2026) · cites this paper as Phelps et al., 2021
DNa02 was identified in the FANC full Drosophila female ventral nerve cord connectome.
“DNa02 was identified in the FANC full Drosophila female ventral nerve cord connectome25 by the lab of Gregory Jefferis.”
From Fine-grained descending control of steering in walking Drosophila (2024) · cites this paper as [25]
Each of the fly's six legs is controlled by 60-70 uniquely identifiable motor neurons.
“Each of the six legs of the fly is controlled by 60–70 uniquely identifiable motor neurons1,5,6 (MNs).”
From Connectomic reconstruction of a female Drosophila ventral nerve cord (2024) · cites this paper as [1]
The FANC electron microscopy volume was previously imaged and a small number of VNC neurons and synapses were manually reconstructed.
“We previously imaged an electron microscopy volume of a Drosophila FANC and manually reconstructed a small number of VNC neurons and synapses1.”
From Connectomic reconstruction of a female Drosophila ventral nerve cord (2024) · cites this paper as [1]
Phelps et al.'s computational analysis, based on NBLAST, identified bundles of neurons with primary neurites running together from soma to axon, but was limited to primary neurites of manually traced motor neurons.
“Then, we used a computational analysis from Phelps et al.1, which relied on the NBLAST algorithm72 to identify bundles, or groups of neurons with primary neurites that run together from the soma to the axon. Phelps et al. limited this analysis to the primary neurites of the manually traced MNs.”
From Connectomic reconstruction of a female Drosophila ventral nerve cord (2024) · cites this paper as [1]
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Self-citation
- Authors
- Phelps, J. S., Hildebrand, D. G. C., Graham, B. J., Kuan, A. T., Thomas, L. A., Nguyen, T. M., Buhmann, J., Azevedo, A. W., Sustar, A., Agrawal, S., Liu, M., Shanny, B. L., Funke, J., Tuthill, J. C., & Lee, W.-C. A.
- Affiliations
- Harvard University (US)
- Howard Hughes Medical Institute (US)
- University of Washington (US)
- Boston Children's Hospital (US)
- Corresponding author
- Lee, Wei-Chung Allen
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2021-02
- Volume
- 184
- Issue
- 3
- Pages
- 759-774.e18
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC8312698
- OpenAlex
- W3120038054
- Cited by
- 265
- References
- 109
- Citations by year
- 2026: 25 · 2025: 44 · 2024: 72 · 2023: 55 · 2022: 30 · 2021: 26 · 2020: 11 · 2019: 2
- FWCI
- 21.17
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Intelligence Advanced Research Projects Activity
- National Institutes of Health
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurobiology and Insect Physiology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Keywords
- ventral nerve cord, Drosophila melanogaster, locomotor circuits, neuronal networks
- MeSH terms
- Aging, Animals, Automation, Drosophila melanogaster, Extremities, Motor Neurons, Peripheral Nerves, Sensory Receptor Cells, Synapses, Microscopy, Electron, Transmission, Connectome
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