Neurolucida explorer software

All collected tissue sections were imaged using a confocal microscope (LSM800; Zeiss, Germany) with the tile-scanning function of Zeiss’s Zen Blue software to cover the entire 2D area of the tissue. All the obtained images were aligned based on the location of the barrel cortex visualized by vGlut2 immunoreactivity (Fig. 2C). The barrel cortex was observed from 3 to 4 consecutive cortical tissue section images. Of these images with distinctive vGlut2 signals, a single image with the brightest BDA injection signal was chosen for further image processing. For the alignment, the barrel cortex was manually contoured, and each section image was rotated up to 8 degrees to fit the barrel cortex into the contour (Fig. 2D). Acquired images in TIF format were imported into Stereo Investigator software (MBF Bioscience, USA). The BDA-traced axons were automatically identified and digitally marked using the “Mark Detected Objects” function embedded in the software (Fig. 2E). The threshold for the object detection function was set to 90.2%. The BDA-marked data file was then exported to Neurolucida Explorer software (MBF Bioscience, USA) and converted into the Cartesian coordinates. The Cartesian coordinates of all the images were aligned based on the coordinates of the injection site (ML: 1.8, AP: 1.5), which was determined as the center of the area with the saturated BDA signal.