Computational protocol: Serotonin-immunoreactivity in the ventral nerve cord of Pycnogonida – support for individually identifiable neurons as ancestral feature of the arthropod nervous system

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Protocol publication

[…] Overview images of complete CNSs were taken with a Zeiss Lumar V12, z-stacks being created with Zeiss AxioVision software (Version 4.7.10) and subsequently merged to a single image with extended depth of field using Heliconsoft Helicon Focus software (Version 4.50).For details, walking leg ganglia were separately scanned with a Leica DM IRE2 confocal laser-scanning microscope equipped with a Leica TCS SP2 AOBS laser-scanning unit, step sizes ranging from 0.4 to 2.0 μm between successive scanning planes, depending on the intended z-resolution. Subsequent analyses were performed with the 3D-reconstruction program ‘Imaris’ (Bitplane AG, Switzerland, version 7.0.0.). Neurites were individually traced in 3D volumes (Surpass mode) as well as in the Section mode, in which all three optical section planes are shown and navigated simultaneously. In order to depict the three-dimensionally extending neurites of individual SL-ir neurons as completely as possible, snapshots taken in Imaris do often not represent single optical sections, but rather Imaris volumes of the scanned image stacks (Maximum intensity projection), to which multiple clipping planes have been applied. In Meridionale sp., the serotonin-immunolabeling resulted in a strong unspecific signal of the neural sheath. To visualize the SL-ir structures within the ganglia more clearly, this unspecific labeling was masked by manual segmentation. Supplementary movies were generated in Imaris (Animation module), being subsequently transformed into MP4-format using the freeware FormatFactory (version 2.96, www.pcfreetime.com). [...] The descriptions of the SL-ir neuron types refer to one body half only, deviations from this are explicitly mentioned. Global contrast and brightness values of some of the images were adjusted using Adobe Photoshop CS3. Images depicting only SL-ir structures were converted into inverted black-and-white images for enhanced contrast. All figures were compiled in Adobe Illustrator CS3. If not stated otherwise in the figure legends, anterior is to the top in all ventral or dorsal aspects and in horizontal sections. In anterior or posterior aspects and transverse sections, dorsal is to the top. […]

Pipeline specifications

Software tools Imaris, Adobe Illustrator
Applications Miscellaneous, Laser scanning microscopy
Chemicals Serotonin