Systematic annotation of a complete adult maleDrosophilanerve cord connectome reveals principles of functional organisation

Abstract

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.

Nested neural circuits generate distinct acoustic signals during Drosophila courtship

“…Having functionally characterized many of the individual song circuit neurons, we next examined their patterns of connectivity with each other and with wing motor neurons 41 within the MANC connectome 40 . This analysis revealed that the eight neuron types required to produce normal amounts of pulse and sine song (pIP10, pMP2, dPR1, dMS9, vMS12, TN1A, dMS2, and vPR9) are highly interconnected to each other and to wing motor neurons (Figures 8A-B, Sup.…”

“…At present, connectomic analysis is also typically limited to very small sample sizes, usually just one, and so we cannot address variability both within and between species. These limitations will eventually be overcome through advances in electron 40,41,67,68,[71][72][73] and light 30 microscopy to provide molecular detail and to allow study of much larger sample sizes.…”

“…Figure 2E, 3E). We identified 11 TN1A neurons in each hemisphere of the MANC EM volume 40 . Our TN1A driver line targets 6.5 ± 1.38 (n=6) neurons, which stochastic labelling 49 confirmed are exclusively of the TN1A subtype (Sup.…”

“…Split-GAL4 enhancer combinations identified in D. melanogaster have been used to target pIP10 in D. yakuba 50 , suggesting that the split-GAL4 reagents we have identified may be useful for studying homologous circuits across Drosophila species. Testing anatomical hypotheses will require comparative connectomics, which should be facilitated by recent advances in EM 40,[65][66][67][68][69][70][71][72] and in expansion microscopy 30 .…”

“…We assess the functions of over 40 distinct cell types by recording and analyzing a total of more than 1800 hours of songs from over 5000 male flies. We then trace the synaptic connections of the identified song neurons in the male VNC connectome (MANC) [40][41][42] . These anatomical and functional data define a highly interconnected core song circuit composed of eight fru and/or dsx-expressing (fru + /dsx + ) cell types.…”