Golgi staining was carried out using the superGolgi Kit (Bioenno Tech, LLC, Santa Ana, CA, USA). Briefly, the mice were anesthetized and the intact brains were removed. The brains were immersed in the impregnation solution. The solution was replaced the next day and the brains were immersed 10 or 12 additional days in the dark, and then transferred to the post-impregnation buffer for 48 h in the dark. Coronal sections (80 μm thick) were cut using a microtome (HM 355S, Thermo Fisher Scientific, Houston, TX, USA). Sections were mounted on adhesive microscope slides using staining solution and post-staining buffer, cleaned, and cover slipped using Permount (FUJIFILM Wako Pure Chemical Corporation). Dendrites of cortical neurons were examined using the Eclipse Ni-U microscope (Nikon Corporation, Tokyo, Japan), and the images were captured with a CCD camera (DS-Fi2, Nikon Corporation, Tokyo, Japan).
For quantification of spines, all images were converted to 8-bit, inverted, and background subtracted using Fiji ImageJ (National Institutes of Health, Bethesda, MD, USA) software, and dendritic spine density was measured by two independent researchers. In each animal, three intact dendritic spines of cortical neurons were selected for more detailed observation.
For quantification of spines, all images were converted to 8-bit, inverted, and background subtracted using Fiji ImageJ (National Institutes of Health, Bethesda, MD, USA) software, and dendritic spine density was measured by two independent researchers. In each animal, three intact dendritic spines of cortical neurons were selected for more detailed observation.