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.

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 theDrosophilacentral 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.

Animal behavior is encoded in neuronal circuits in the brain. To elucidate the function of these circuits, it is necessary to identify, record from and manipulate networks of connected neurons. Here we present BAcTrace ( B otulinum Ac tivated Tracer ), a genetically encoded, retro-grade, transsynaptic labelling system. BAcTrace is based on C. botulinum neurotoxin A, Botox, which we have engineered to travel retrogradely between neurons to activate an otherwise silent transcription factor. We validate BAcTrace at three neuronal connections in the Drosophila olfactory system. We show that BAcTrace-mediated labeling allows electrophysiological recordings of connected neurons. Finally, in a challenging circuit with highly divergent connections, BAcTrace correctly identifies 12 out of 16 connections, which were previously observed by electron microscopy.

Sex pheromones are key social signals in most animals. In Drosophila a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA) that promotes female courtship while repelling males. Here we show that flies use separate cVA processing streams to extract qualitative and positional information. cVA olfactory neurons are sensitive to concentration differences in a 5 mm range around a male. Second-order projection neurons detect inter-antennal differences in cVA concentration, encoding the angular position of a male. We identify a circuit mechanism increasing left-right contrast through an interneuron which provides contralateral inhibition. At the third layer of the circuit we identify neurons with distinct response properties and sensory integration motifs. One population is selectively tuned to an approaching male with speed-dependent responses. A second population responds tonically to a male's presence and controls female mating decisions. A third population integrates a male taste cue with cVA; only a simultaneous presentation of both signals promotes female mating via this pathway. Thus the olfactory system generates a range of complex percepts in discrete populations of central neurons that allow the expression of appropriate behaviors depending on context. Such separation of olfactory features resembles the mammalian what and where visual streams.

11New tools and techniques have enabled many key advances in our understanding of the brain. To elucidate circuit function, it is necessary to identify, record from and manipulate networks of connected neurons. Here we present BAcTrace (Botulinum Activated Tracer), the first fully genetically encoded, retrograde, transsynaptic labelling system. BAcTrace is based on C. botulinum neurotoxin A, Botox, which we have engineered to act as a Trojan horse that jumps retrogradely between neurons to activate an otherwise silent transcription factor. We validate BAcTrace at three connections in the Drosophila olfactory system and show that it enables electrophysiological recordings of connected neurons. Finally, in a challenging circuit with highly divergent connections, we used Electron Microscopy connectomics to show that BAcTrace correctly identifies 12 out of 16 connections.The development of genetic tools to elucidate connectivity and manipulate neurons and circuits has 13 been key to advancing our understanding of how the brain works. Increasingly these tools are being 14 used to study diseases of the nervous system and develop effective treatments [31, 57]. 15In the context of circuit research, the ability to identify and manipulate pre-or post-synaptic cells to 16 neurons of interest is of crucial importance. For example, if genetic drivers are available for sensory 17 neurons in the skin then one might want to label downstream, post-synaptic cells in the nerve cord. 18Conversely, when studying motor circuits, genetic drivers for motor neurons might be available and 19 revealing upstream, pre-synaptic cells will be appropriate. Tools to label downstream neurons (e.g. for 20 "walking" from sensory input towards motor-outputs as in the first example) are called anterograde, 21 while retrograde tools reveal the input neurons to a given population. 22Drosophila melanogaster is a key model organisms to study the genetic and circuit basis of animal 23 behaviour (e.g. see [62, 15]). The fly has a rich behavioural repertoire encoded in a relatively small 24 nervous system. This numerical simplicity is paired with extensive collections of genetic reagents, 25 both to investigate gene function (e.g. mutant and RNAi collections) and to label and manipulate 26 most neuronal classes (using the orthogonal expression systems Gal4, LexA, QF and their split versions 27 [4, 45, 30, 58]). While these reagents offer excellent genetic access to neurons, until recently the 28 fly lacked tools to map synaptic connections between neurons. This has recently changed with the 29 development of electron microscopy methods to map connections in larval [38] and adult [63] brains. 30 Furthermore, two genetically encoded systems for anterograde tracing: trans-Tango [54] and TRACT 31 [22] have recently been developed. Despite these important additions to the experimental toolbox, a 32 retrograde labelling system is still missing. Rabies virus and its modifications constitute the most notable 33 examples of retrograde transsynaptic tools [31]. While...