Quantification of Corticospinal Tract in Mouse Brains

Fixed 129SvPas/C57Bl6-Thy1-eYFP brains were cut into 40-µm slices using a cryo-microtome (Microm, H560S); slices were mounted on Superfrost plus slides (Menzel-Gläser). Surface quantifications were performed on coronal slices. Epifluorescence of sections was digitized using an Axioscop microscope (Zeiss) equipped with a 5x dry objective and an EMCCD Quantum camera controlled by Metamorph software (Roper Scientific). ROIs were manually drawn and surface areas were calculated using ImageJ software. Since the cerebral volume for WT and KO mice was not the same^{21 (link)}, the area of the corticospinal tract was quantified on coronal brain sections selected based on morphometric landmarks. Thus, slice B corresponded to the end of the dentate gyrus (Bregma: −1.34 mm); slice C corresponded to the start of the habenular commissure (Bregma: −2.30mm); Slice D corresponded to the end of cerebral peduncle lateralization and the start of the rubrospinal tract (Bregma: −4.04 mm); and slice E corresponded to the 10^th slice before the appearance of pyramidal tract decussation (Bregma: −7.56 mm). Epifluorescence for the pyramidal tract was further analyzed on sagittal sections using a confocal microscope (LSM 710, Zeiss), or in whole-mount brainstems using a stereo-microscope (Olympus SZX12).