In most animals, a relatively small number of descending neurons (DNs) connect higher brain centers in the animal’s head to motor neurons (MNs) in the nerve cord of the animal’s body that effect movement of the limbs. To understand how brain signals generate behavior, it is critical to understand how these descending pathways are organized onto the body MNs. In the fly, Drosophila melanogaster, MNs controlling muscles in the leg, wing, and other motor systems reside in a ventral nerve cord (VNC), analogous to the mammalian spinal cord. In companion papers, we introduced a densely-reconstructed connectome of the Drosophila Male Adult Nerve Cord (MANC, Takemura et al., 2023), including cell type and developmental lineage annotation (Marin et al., 2023), which provides complete VNC connectivity at synaptic resolution. Here, we present a first look at the organization of the VNC networks connecting DNs to MNs based on this new connectome information. We proofread and curated all DNs and MNs to ensure accuracy and reliability, then systematically matched DN axon terminals and MN dendrites with light microscopy data to link their VNC morphology with their brain inputs or muscle targets. We report both broad organizational patterns of the entire network and fine-scale analysis of selected circuits of interest. We discover that direct DN-MN connections are infrequent and identify communities of intrinsic neurons linked to control of different motor systems, including putative ventral circuits for walking, dorsal circuits for flight steering and power generation, and intermediate circuits in the lower tectulum for coordinated action of wings and legs. Our analysis generates hypotheses for future functional experiments and, together with the MANC connectome, empowers others to investigate these and other circuits of the Drosophila ventral nerve cord in richer mechanistic detail.

Animal behavior is principally expressed through neural control of muscles. Therefore understanding how the brain controls behavior requires mapping neuronal circuits all the way to motor neurons. We have previously established technology to collect large-volume electron microscopy data sets of neural tissue and fully reconstruct the morphology of the neurons and their chemical synaptic connections throughout the volume. Using these tools we generated a dense wiring diagram, or connectome, for a large portion of the Drosophila central brain. However, in most animals, including the fly, the majority of motor neurons are located outside the brain in a neural center closer to the body, i.e. the mammalian spinal cord or insect ventral nerve cord (VNC). In this paper, we extend our effort to map full neural circuits for behavior by generating a connectome of the VNC of a male fly.

Our companion paper (Takemura et al., 2023) introduces the first completely proofread connectome of the nerve cord of an animal that can walk or fly. The base connectome consists of neuronal morphologies and the connections between them. However, in order to efficiently navigate and understand this connectome, it is crucial to have a system of annotations that systematically categorises and names neurons, linking them to the existing literature. In this paper we describe the comprehensive annotation of the VNC connectome, first by a system of hierarchical coarse annotations, then by grouping left-right and serially homologous neurons and eventually by defining systematic cell types for the intrinsic interneurons and sensory neurons of the VNC; descending and motor neurons are typed in (Cheong et al., 2023). We assign a sensory modality to over 5000 sensory neurons, cluster them by connectivity, and identify serially homologous cell types and a layered organisation likely corresponding to peripheral topography. We identify the developmental neuroblast of origin of the large majority of VNC neurons and confirm that (in most cases) all secondary neurons of each hemilineage express a single neurotransmitter. Neuroblast hemilineages are serially repeated along the segments of the nerve cord and generally exhibit consistent hemilineage-to-hemilineage connectivity across neuromeres, supporting the idea that hemilineages are a major organisational feature of the VNC. We also find that more than a third of individual neurons belong to serially homologous cell types, which were crucial for identifying motor neurons and sensory neurons across leg neuropils. Categorising interneurons by their neuropil innervation patterns provides an additional organisation axis. Over half of the intrinsic neurons of the VNC appear dedicated to the legs, with the majority restricted to single leg neuropils; in contrast, inhibitory interneurons connecting different leg neuropils, especially those crossing the midline, appear rarer than anticipated by standard models of locomotor circuitry. Our annotations are being released as part of the neuprint.janelia.org web application and also serve as the basis of programmatic analysis of the connectome through dedicated tools that we describe in this paper.