3D Brain Reconstruction and Single Neuron Tracing

The criteria for selecting suitable preparations for 3D reconstruction were the least damage in regions of interest and sufficient contrast through the entire stack. Out of totally nine immuno-stained male brains, three were selected to construct either full brains or all neuropils of the central brain. Raw confocal stacks were aligned and stitched together using FIJI (Preibisch et al., 2009) and resampled to 1.5 × 1.5 × 1.5 μm voxel size in Amira (Amira 5.3; Thermo Fisher, Visualization Science Group). These complete image stacks were then utilized to carry out image segmentation in Amira. We manually labeled key cross sections of each neuropil of interest in all three spatial planes and then used the wrap-tool in Amira to obtain full neuropil volumes. This process yielded segmented image stacks containing all major neuropils of the moth brain. A surface model of the segmented image stacks was produced in Amira. These were either visualized in Amira or exported as obj-files and uploaded to the Insect Brain Database¹ for visualization.

Five stained neurons from three preparations were traced manually using the SkeletonTree plugin in Amira (Schmitt et al., 2004; Evers et al., 2005). Based on background fluorescence of the neuron channel, neuropils close to the traced neurons were segmented in each brain preparations as described above.