Computational protocol: Vision in two cyprinid fish: implications for collective behavior

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Protocol publication

[…] We extracted and wholemounted the retinas of 3 adult zebrafish and 3 adult golden shiners. Following euthanasia via rapid cooling (), we submerged the head of the animal in Davidson’s Fixative for 3–5 h. While the eye remained attached to head by the optic nerve, we used spring scissors to remove the cornea, lens and vitreous humor. A dorsal cut was made to the retina before the eyecup was detached to ensure the correct orientation in later steps. We separated the retina from the eyecup by peeling away the sclera with forceps. The retina was then subjected to a (3%) bleaching solution until transparent to allow for the visualization of the ganglion cells. Following bleaching, 3–4 peripheral cuts were made to allow the retina to lie flat on a gelatinized glass slide. We followed the procedures described in to fix and stain the retina using a series of dehydrating and rehydrating steps in combination with 0.1% cresyl violet. In addition, we photographed images of the wholemounted retina and recorded area and perimeter measurements to account for retina shrinkage before and after staining using ImageJ ( Average retina shrinkage values for the zebrafish and golden shiner were 0.07 ± 0.05 and 0.13 ± 0.07 respectively (mean ± SE).Following staining, the retinas were viewed under an Olympus BX51 microscope. We traced the perimeter of the retinal periphery and optic nerve head with Stereo Investigator v.10 (MBF Bioscience, Williston, Vermont, USA) and then applied a sampling grid with a counting frame of 50 µm2 × 50 µm2 for a total of approximately 200 total sampling sites per retina. We then counted the total number of observed retinal ganglion cells at each of the sampling sites using ImageJ ( Ganglion cells were distinguished from amacrine and glial cells according to previously established morphological characteristics (; ; ). We omitted counting sites where the retina appeared damaged or the cell visibility was poor. For the zebrafish, we counted 150 ± 54.9 sites per retina; while for the golden shiner we counted 112 ± 15.7 sites (mean ± SE). The Schaeffer coefficient of error for the zebrafish and golden shiner was 0.006 ± 0.0015 and 0.003 ± 0.0002 respectively (). Topographic maps were generating using the R program “one cell map V8 svg version.R” (). This program constructs isodensity maps from regions of localized cell density within a defined retinal outline created with Adobe Illustrator. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Danio rerio, Notemigonus crysoleucas