Complete connectomic reconstruction of olfactory projection neurons in the fly brain

Abstract

Nervous systems contain sensory neurons, local neurons, projection neurons and motor neurons. To understand how these building blocks form whole circuits, we must distil these broad classes into neuronal cell types and describe their network connectivity. Using an electron micrograph dataset for an entire Drosophila melanogaster brain, we reconstruct the first complete inventory of olfactory projections connecting the antennal lobe, the insect analogue of the mammalian olfactory bulb, to higher-order brain regions in an adult animal brain. We then connect this inventory to extant data in the literature, providing synaptic-resolution 'holotypes' both for heavily investigated and previously unknown cell types. Projection neurons are approximately twice as numerous as reported by light level studies; cell types are stereotyped, but not identical, in cell and synapse numbers between brain hemispheres. The lateral horn, the insect analogue of the mammalian cortical amygdala, is the main target for this olfactory information and has been shown to guide innate behaviour. Here, we find new connectivity motifs, including: axo-axonic connectivity between projection neurons; feedback and lateral inhibition of these axons by local neurons; and the convergence of different inputs, including non-olfactory inputs and memory-related feedback onto lateral horn neurons. This differs from the configuration of the second most prominent target for olfactory projection neurons: the mushroom body calyx, the insect analogue of the mammalian piriform cortex and a centre for associative memory. Our work provides a complete neuroanatomical platform for future studies of the adult Drosophila olfactory system. Highlights• First complete parts list for second-order neurons of an adult olfactory system • Quantification of left-right stereotypy in cell and synapse number • Axo-axonic connections form hierarchical communities in the lateral horn • Local neurons and memory-related feedback target projection neuron axons 149 197 346 Figure 1: Second-order olfactory projection neurons. A Layout of the mammalian olfactory system. B The olfactory system in fruit flies shares similarities with that of mammals. C Second-order olfactory projection neurons (PNs) in all antennal lobe tracts (ALT) were reconstructed from a serial section transmission electron microscopy (ssTEM) volume of an entire fly brain. Scale bar represents 1 micron. D Exemplary sparse (left) and broad (right) PN. Dendrites used for classification in blue. E PNs were classified as uni-(uPN) or multiglomerular (mPN) projection neurons based on the sparseness of their glomerular innervation (see also Figure S1). F PN counts by class and neurotransmitter. G Comparison of right-(RHS) vs left-hand-side (LHS) cell counts for 58 of the 78 uPN types. H Scaling of the olfactory system from larval to adult D. melanogaster. Bates, Schlegel et al. 2 / 31

Connectomics analysis reveals first, second, and third order thermosensory and hygrosensory neurons in the adultDrosophilabrain

“…Skeleton fragments that had been automatically segmented with the floodfilling technique [52] were manually joined in a separate CATMAID instance, imported into the manual CATMAID instance, and merged with manually traced partial skeletons where possible (https://github.com/flyconnectome/fafbseg-py). Skeletons were either traced to identification (with soma, backbone, and main branches) or to completion (with all twigs, including pre-and postsynapses, and fully reviewed [36]. Synapses were annotated based on previously described criteria: thick, dark active zone, presynaptic membrane specialisations (T-bars, vesicles), and a synaptic cleft [55].…”

“…The dendrites of lACA-associated KCs on the RHS of FAFB were completed in CATMAID, and their postsynapses were manually associated with upstream neurons -mainly RHS or bilaterally symmetric antennal lobe PNs, which had already been traced to identification in this dataset [32,36]. Analysis of KC inputs was performed in R with a customised script, utilising the nat and elmr packages from the natverse.…”

“…This was repeated 1000 times to generate an average number of expected inputs for the population. Inputs from each glomerulus were assigned a putative valence based on the olfactory literature [36]. Ir21a-T2A-Gal4 and Ir68a-T2A-Gal4 where generated using techniques previously described [60].…”

“…Based on their primary soma tracts, the VP PNs belonged to seven distinct neuroblast lineages: AL anterodorsal (ad), lateral (l), ventral (v), or lateroventral (lv) [35], or the previously unidentified inferior lateral (il), inferior ventral (iv), or inferior lateroventral (ilv) [36]. The lateral lineage generated the most VP PNs [37] and exhibited the most diversity in axon tract (Table 1).…”

Automatic Detection of Synaptic Partners in a Whole-BrainDrosophilaEM Dataset

“…A second dataset P LH contains 389 neurons (including the three projection neurons from O LH) with complete known connectivity inside the lateral horn (24,846 connections). The three olfactory projection neurons are published byHuoviala et al (2018), remaining data is published byBates et al (2020).I O EB contains 27 neurons, for which all 61,280 incoming and outgoing connections have been annotated inside the EB. Data is published byTurner-Evans et al (2019).I O PB contains the same 27 neurons as in I O EB but neuron parts with all their 14,779 incoming and outgoing connections are located in PB.…”