2.8 Golgi staining
The FD Rapid Golgi-Staining Kit (FD NeuroTechnologies, Columbia, MD, USA) was used to reveal the density of dendritic spines in pyramidal neurons in the PrL and IL. Brains were immersed in Golgi-staining impregnation solution for 2 weeks. Sections were cut at 150 µm on a cryostat at -20°C to -22°C. The spines on secondary or tertiary dendrites of pyramidal neurons were calculated at a dendritic segment length of approximately 50 µm. At least 3 dendrites per rat were traced, and a total of 6 rats per group were counted. The images were captured under Nikon Eclipse Ci-L microscope (Nikon, Tokyo, Japan) using DP controller software with 100×A/1.25 oil immersion lens. The density of dendritic spines and the dendritic spine morphologies in the PrL and IL were analysed using ImageJ software. Dendritic spine density was calculated as the total number of spines per 10 µm length of branch. Dendritic spine morphologies were classified into 4 main types: thin, filopodia, mushroom and stubby(Tackenberg, Ghori et al., 2009). The thin type has a narrow neck with elongated protrusion; the filopodia type was identified as long, thin structures; the mushroom type has a large irregular head with a neck diameter smaller than the head diameter; the stubby type has no obvious constriction between the protrusion and attachment to the neck. The proportion of each type was quantified as ([spine number with each type/total spine number] ×100).