Golgi–Cox staining

We used a commercially available Golgi–Cox staining system, the FD Rapid Golgi Staining kit, which provides an adequate number of well-impregnated neurons with clearly visible spines. Briefly, mice were deeply anesthetized with ketamine/xylazine cocktail until lack of response to toe pinch was recorded. The brain was quickly collected from the skull and rinsed with Milli-Q water to remove blood from the surface. Impregnation was performed following the manufacturer’s instructions. Further processing of samples was done at FD Neurotechnologies. Images of pyramidal neurons from the CA1 area of the hippocampus were collected on 100 μm-thick slices using a Zeiss 780 confocal laser-scanning microscope. In order to detect Golgi–Cox staining, the microscope was set up in transmission mode, and 488 nm wavelength laser was used. Confocal images were obtained using a Plan-Neofluar 63× water (1.3 numerical aperture) objective. Each frame was acquired eight times and then averaged to obtain noise-free images. Spine density was measured by a person blinded to genotype and using Fiji imaging analysis software (http://fiji.sc/Fiji).