Computational protocol: The Non-Lemniscal Auditory Cortex in Ferrets: Convergence of Corticotectal Inputs in the Superior Colliculus

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Protocol publication

[…] Histological analysis was carried out and photomicrographs were taken with a Leica DMR microscope (Leica Microsystems, Heerbrugg, Switzerland) and a digital camera (Microfire™, Olympus America Inc, Center Valley, PA, USA). Histological reconstructions and unbiased stereological estimates were performed using Neurolucida and StereoInvestigator software (version 8, MBF Bioscience, MicroBrightField Inc., Williston, VT, USA). Three dimensional reconstructions from the sections were performed using the 3D module in Neuroexplorer (MBF Neuroscience). One set of serial sections (1 every 300 μm) at the level of the SC or at the level of the auditory cortex was plotted in StereoInvestigator using a Cartesian coordinate system. The sections were aligned using blood vessels and outlines of adjacent sections. Statistical analysis was carried out using SPSS software (SPSS Inc., Chicago, IL, USA).To estimate the relationship between the labeled terminals and the different layers of the SC, we reconstructed the position of the terminals and of the intermediate and deep gray layers in this nucleus using the center of the fourth ventricle at the most posterior section in which the SC is present as a reference point, i.e., the point that marks the 0,0,0 origin of our Cartesian coordinate system. We used three independent metrics to compare the distribution of the terminals in the deep SC cellular layers across animals. The first metric compared the dispersion and the clustering indexes. The dispersion index was calculated as the ratio of the area of labeling in the SC layers normalized by the area of the injection site in the cortex. Those areas (A in the following equation) were calculated by drawing a polygon around the labeled terminals, within the limits of the SGI and SGP, and around the limits of the injection sites (x, y, and z represent the three spatial coordinates):The clustering index (CI) is the mean distance between a terminal and its closest neighbor in the same layerTherefore, larger values in the dispersion and clustering indexes indicate greater divergence in the projection and vice versa.The second metric relates the distribution of the terminals to the spatial extent of each SC layer by comparing the locations of the centroids (Cx and Cy) for each in every section examined. The centroids were defined by the intersection of all the hyperplanes of symmetry of each terminal distribution or SC layer according to the following equations. This comparison provides a measure of how homogenous the terminal labeling is within each layer.To further analyze the distribution of the cortical terminals within the SC, the intermediate and deep layers were divided into four equal quadrants based on their centroid coordinates and the anteroposterior axis. The number of terminals in each quadrant was compared across animals. The density of terminals in the SC was calculated by dividing the total number of terminals (estimated using the fractionator after counting one in every six sections without employing counting frames) by the volume of each layer. The volumes of the SGI and SGP were calculated by the Cavalieri method (Gundersen, ). This involved multiplying the area of one in every six sections along the anteroposterior axis of the SC by the distance between them. The small size of the terminals relative to the thickness of the sections prevented any possible double counting.Analysis of the morphological features of the labeled terminals was performed using optical microscopy. We considered every swelling in a labeled fiber as an “en passant” bouton and every swelling at the end of a labeled fiber as an end-terminal bouton. Terminals were classified according to their morphology following the same criteria used by Fuentes-Santamaria et al. () in the cat SC. […]

Pipeline specifications

Software tools Neurolucida, NeuroExplorer
Application Neurophysiology analysis
Organisms Mustela putorius furo