Computational protocol: Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems

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Protocol publication

[…] Immunofluorescence and immunohistochemistry were carried out as described using rabbit anti-tyrosine hydroxylase antibody (1:500 (ref. )) and chicken anti-GFP (1:400, Molecular Probes) or mouse anti-GFP antibodies (1:1,000, Roche). Alexa555- or Alexa594-conjugated goat anti-rabbit IgG (1:2,000 and 1:1,000, respectively, Molecular Probes) and Alexa488-conjugated goat anti-chicken IgG or anti-mouse IgG (1:600 and 1:1,000 respectively, Molecular Probes) were used as secondary antibodies. Embryos were mounted with 80% glycerol/1.2% low-melting agarose/PBS.Confocal Z stacks were recorded with a Zeiss LSM 510 laser-scanning confocal microscope with pinhole diameters set to one airy unit for Alexa555 and Alexa488-labelled embryos using the 488- and 561-nm laser lines. Embryos stained with Alexa594 and Alexa488 were examined under Zeiss LSM 510 NLO with pinhole diameters between 360–520 μm using the two-photon 770- nm laser line. All stacks were recorded with a 25× NA 0.8 multi-immersion lens (LD-LCI Plan-Apochromat) and glycerol immersion medium. Stack sizes are 1,024×1,024 pixel in xy direction, with the number of z planes depending on the thickness of the sample. The voxel sizes used were 0.5 μm×0.5 μm×1.3 μm in the z direction. Pixel times of 1.26–3.20 μs were used.To better visualize neuronal projections in the context of the anatomical structures, differential interference contrast microscopy was used (). Following confocal analysis, embryos of interest were incubated overnight in 1% goat serum/PBST containing DSB-X biotin-conjugated goat anti-chicken IgG (1:1,000, Molecular Probes), processed using biotinylated HRP and avidin reagents (Vectastain ABC HRP kit), and stained with the chromogen diaminobenzidine. Images were documented with DIC Axioscope2/AxioCam MRc (Zeiss) and processed with ImageJ (http://rsbweb.nih.gov/ij/). [...] For far-projecting neurons, adjacent whole-brain image stacks were recorded. To enhance the visualization of GFP-tagged somata and their projections, we sought a simple solution to isolate them from nonspecific signals. In the whole-larvae preparations, immunofluorescence noise, auto-fluorescence (for example, some pigment cells), as well as other GFP expressing cells build up significant nonspecific signals when visualizations of image stacks are prepared. We wanted to avoid established noise-reduction approaches including thresholding or despeckling, given that fine CA processes in our stacks often had cross-section areas of very few pixels only. Therefore, we developed an image-processing pipeline for interactive in silico feature extraction, which is amenable to automated batch operation and includes a quality control element that allows independent and objective verification of 3D objects ().The data set for a single brain recording was processed as follows (): Maximum intensity projections (MIP) in dorsal and lateral directions and MIP movies of the revolving 3D brain image were created for quick visualization. The user proceeded to draw the dorsal and lateral segmentation masks over the MIPs using our ImageJ HDF5 plugin (http://lmb.informatik.uni-freiburg.de/lmbsoft/imagej_plugins/). The 3D intersection M of the 2D lateral L and dorsal D masks was computed as M(x,y,z)=D(x,y) L(x,z), where M, D and L are scalar fields containing 1 for object pixels and 0 for background pixels. The resulting 3D mask was applied to the green channel, thereby allowing the segmentation of labelled neurons within a single brain and disregarding nonspecific signals and background (). For quality control procedures as well as comparison to other projection tracking approaches, see and . All figures were assembled using Adobe Photoshop CS2 or Adobe Illustrator CS4. Brightness and contrast levels were adjusted in Photoshop to ensure that fine axonal and dendritic processes appear in print. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Danio rerio, Homo sapiens