Mice (8-10 weeks) were anesthetized and perfused with a solution containing 2% paraformaldehyde and 1.5% glutaraldehyde, as described (Marker et al. 2005). Coronal sections (60 μm) were cut through the level of the NAcc using a vibrating microtome. After several washes in PB, sections were post-fixed with 1% osmium tetraoxide in PB and block-stained with 1% uranyl acetate in distilled water. Sections were then dehydrated in ascending series of ethanol (to 100%) followed by propylene oxide and flat-embedded on glass slides in Durcupan (Sigma; St. Louis, MO). For quantitative analysis of the density of spines/synapses in the NAcc, sampling fields were chosen by using the systematic random sampling method (Geinisman et al. 1996). After collection of semi-thin sections and determination of sample sites, serial ultrathin sections (80 nm thick) of the neuropil containing the sampling fields were collected from three different parts of the NAcc of three different animals. Sections were placed on slot grids and stained with Reynolds’ lead citrate.
