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).