Golgi staining was performed with the FD Rapid Golgistain™ Kit (FD Neurotechnologies, MD) according to the manufacturer's instructions. In brief, freshly dissected brain was kept in impregnation solution A and B containing potassium dichromate and chromate for two weeks at room temperature. After being replaced with solution C for 48 hours at 4°C, brain tissues were sectioned into 150 µm and fixed to gelatine coated slides. The mixture of solution D, E and distilled water (1∶1∶2 ratio) were used for staining pyramidal neurons for 10 mins, followed by hydration in 50%, 75%, 95% ethanol and absolute ethanol. The brain sections were finally cleared in xylene and covered by coverslip in permount. Five neurons from 150 um-thick sections were analyzed using Neurolucida (MicroBrightField, USA) and selected according to the method described by Woolley et al. [23] (link). Only neurons located in the CA3 region of the dorsal hippocampus were selected for analysis. The selected neurons have to be relatively isolated from neighbouring impregnated neurons to avoid interference with analysis. Cells bodies should be located in the middle part of the section thickness so as to minimize the cut of branch segments. Also, the neurons should be consistently and darkly impregnated along the entire extent of all dendrites. The spine density was estimated by randomly selecting high-magnification tracing of a >10 µm-long terminal segment of the basal/apical dendritic branch. Three to five tertiary apical dendrites and basal dendrites with at least one branch point were selected for counting. The visible spines along the branch segment were counted and data were expressed as number/10 µm.
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