Neuron reconstructions are based on manual skeleton tracing of neuronal arbors and annotation of synapses from image stacks in CATMAID (http://www.catmaid.org ) as described in Schneider-Mizell et al. (2016) (link). All neurons included in analyses are reconstructed by at least two team members, an initial tracer and a subsequent proofreader who corroborates the tracer’s work. In the event that any tracer or proofreader encounters ambiguous features (neural processes or synapses that are not identifiable with high confidence), they consult other tracers and proofreaders to determine the validity of said features, climbing the experience ladder up to expert tracers as needed. If any feature remains ambiguous after scrutiny by an expert tracer, then said feature is not included in the neural reconstruction and/or flagged to be excluded from analyses. During the proofreading phase, the proofreader and tracer iteratively consult each other until each neuron is deemed complete per the specific tracing protocol to which it belongs. An assignment of completion does not necessarily entail that an entire neuron’s processes and synapses have been reconstructed (see Tracing to classification and Tracing to completion). We traced 114 PNs, the APL, two MB-C1s, MB-CP1, and two MB-CP2 neurons to classification (120 neurons in total). We also traced the calyx sub-arbors of the 15 KCs to completion, and their remaining sub-arbors to morphological, but not synaptic, completion. The total cable length of the neurons above is 206.6 mm.
The criteria to identify a chemical synapse include at least three of the four following features, with the first as an absolute requirement: 1) an active zone with vesicles; 2) presynaptic specializations such as a ribbon or T-bar with or without a platform; 3) synaptic clefts; 4) postsynaptic membrane specializations such as postsynaptic densities (PSDs). In flies, PSDs are variable, clearer at postsynaptic sites of KCs in a microglomerulus but often subtle, unclear, or absent in other atypical synaptic contacts (Prokop and Meinertzhagen, 2006 (link)). In the absence of clear PSDs, we marked all cells with membranes that have unobstructed access to a clearly visible synaptic cleft as postsynaptic. We did not attempt to identify electrical synapses (gap junctions), since they are unlikely to be resolved at the resolution of this dataset.
Very rarely, aberrant neurons were found in the dataset. For example, two PNs with a piece of fused cell membrane were discovered in our tracing. It is unknown what factors might cause this, but cell membrane pathologies resultant from EM fixation protocols have been observed (Kopek et al., 2017 (link)). Overall, however, the ultrastructural quality of the whole brain was excellent.
The criteria to identify a chemical synapse include at least three of the four following features, with the first as an absolute requirement: 1) an active zone with vesicles; 2) presynaptic specializations such as a ribbon or T-bar with or without a platform; 3) synaptic clefts; 4) postsynaptic membrane specializations such as postsynaptic densities (PSDs). In flies, PSDs are variable, clearer at postsynaptic sites of KCs in a microglomerulus but often subtle, unclear, or absent in other atypical synaptic contacts (Prokop and Meinertzhagen, 2006 (link)). In the absence of clear PSDs, we marked all cells with membranes that have unobstructed access to a clearly visible synaptic cleft as postsynaptic. We did not attempt to identify electrical synapses (gap junctions), since they are unlikely to be resolved at the resolution of this dataset.
Very rarely, aberrant neurons were found in the dataset. For example, two PNs with a piece of fused cell membrane were discovered in our tracing. It is unknown what factors might cause this, but cell membrane pathologies resultant from EM fixation protocols have been observed (Kopek et al., 2017 (link)). Overall, however, the ultrastructural quality of the whole brain was excellent.
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