Wide field images were taken using an Olympus BX61WI fluorescence microscope (Olympus, Japan) with a Volocity software (Quorum Technologies) or a Zeiss Axio Imager Z2 (Zeiss, Germany) with a colibri LED 7 and Zen blue software (Zeiss, Germany). Some images were taken using a Zeiss LSM700 confocal microscope (BioVis facility, Uppsala University). Brightness and contrast were adjusted in ImageJ, equally for the whole image and without obscuring any data. Images of brainstem were stitched using the Pairwise Stitching in ImageJ (ImageJ, RRID:SCR_003070) (Preibisch et al., 2009 (link)). Cell counts were done manually with ImageJ's Cell counter plugin (Schneider et al., 2012 (link)). For estimation of the total number of traced cells in the spinal cord, cells were identified and counted using an ImageJ macro based on signal intensity and size. A selection of automatically counted images was re‐counted manually, and the macro was found to identify 50–80% of cells.
Brain regions were identified based on the Allen Brain atlas and The Mouse Brain in Stereotaxic Coordinates (MBSC) (Franklin & Paxinos, 2008 ; Lein et al., 2007 (link)). Abbreviations followed the MBSC.
Brain regions were identified based on the Allen Brain atlas and The Mouse Brain in Stereotaxic Coordinates (MBSC) (Franklin & Paxinos, 2008 ; Lein et al., 2007 (link)). Abbreviations followed the MBSC.
