Computational protocol: The Regulation of Corticofugal Fiber Targeting by Retinal Inputs

Similar protocols

Protocol publication

[…] The ingrowth of fluorescent fibers to the dLGN and superficial gray layer of the SC was quantified by analyzing the intensity profiles of pixels within the dLGN or superficial gray layer of the SC on fluorescent microscope images taken on a Leica DMR fluorescence microscope with a DC500 camera and Leica Firecam software (Leica Microsystems, Milton Keynes, UK). Images from the control and manipulated hemisphere were taken with identical microscope and camera settings. In addition, image intensity was standardized between brains in ImageJ () by measuring fluorescence intensity within ventral posterior-medial nucleus (VPM) on thalamic sections or periaqueductal gray on tectal sections. Any images, in which fluorescence intensity departed from the average was adjusted using the brightness tool in ImageJ.The images acquired contralateral to the manipulated (removed/ injected) eye were compared with the images ipsilateral to the manipulated eye of the same brain (as an intra-animal control). Analysis was done blind to experimental condition. The region of interest for analysis was selected by the following criteria—the dLGN: the lateral border: “intralaminar” GFP or tdTomato bundle between dLGN and vLGN; ventral border: thick GFP/tdTomato-positive bundle between the dLGN and VPM; medial border: curved GFP/tdTomato bundle between the dLGN and the posterior thalamic nucleus (Po); the dorsal border: the edge between the dLGN tissue and the lateral ventricle. In the ventral border, the line selection tool was drawn through the mid-point of the GFP/tdTomato bundle. In the lateral corner of the dLGN: the dorsal, lateral, and ventral edges of the dLGN were defined as above. To outline just the lateral half of the dLGN, the “medial” outline was done halfway between the lateral edge of the nucleus and the medial edge of the nucleus. In the superficial gray layer: the ventral border: the border between the superficial gray layer and the optic nerve layer as assessed by the density of tdTomato-positive fibers (which are in thicker bundles in the optic nerve layer). The dorsal edge of the SC was used as the dorsal border of the superficial gray layer. The medial edge of the SC was used as the medial edge of the superficial gray layer. The lateral edge the superficial gray is where the dorsal edge and ventral edges meet.In the selected area, the gray-scale intensity value of every pixel was captured using the Record Measurements tool in Adobe Photoshop CS5 (Adobe Systems Inc., CA, USA). This quantifies the number of pixels at every gray-scale intensity value (range 0–255). Using these data, the frequency of pixels above the threshold was calculated for every intensity threshold in the range, and normalized by the number of pixels within the area of the dLGN. This measures the proportion of pixels within the dLGN which are above any gray-scale intensity threshold.Statistical comparisons were performed at every 10th pixel intensity threshold (10–120) for the thresholds above which the proportion of pixels drops from 1 and below which the proportion of pixels is above 0 in GraphPadPrism (GraphPad Software, CA, USA). Control and enucleated dLGN were compared using one-tailed, paired t-test. In all graphs, * = significant at P = 0.05, ** = significant at P = 0.005, *** = significant at P < 0.0005. […]

Pipeline specifications

Software tools ImageJ, GraphPad Prism
Applications Miscellaneous, Microscopic phenotype analysis
Chemicals Retinaldehyde