Synaptoneurosomes Isolation from Rat NAc

Rats were decapitated and bilateral NAc tissue (primarily core) was rapidly dissected from a 2 mm coronal slice prepared with a brain matrix (ASI Instruments). Immediately following dissection, synaptoneurosomes were prepared according to published protocols (Most et al., 2015 (link); Workman et al., 2015 (link); Werner et al., 2018 (link)). NAc punches were homogenized in 500 μl of homogenization buffer [HB; 20 mm HEPES, 0.5 mm EGTA, 1× Proteasome Inhibitor Cocktail Set 1 (Millipore)]. Homogenates were passed through a 100-μm-pore filter and then through a 5-μm-pore filter (Millipore; both filters were prewashed with HB). After homogenates were passed through each filter, filters were washed with 50 μl of HB, and the washes were added to homogenates to maximize yield. Homogenates were then centrifuged at 14,000 × g for 20 min at 4°C. The pellet, which contains the synaptoneurosomes, was frozen on dry ice, stored at −80°C, and ultimately lysed in lysis buffer [0.605 × g Tris-HCl, 0.25 × g sodium deoxycholate, 0.876 × g NaCl, 1 μg/ml PMSF, 5 ml of 20% SDS, and 1× Protease Inhibitor Cocktail Set 1 (Millipore) in 100 ml of dH₂O] for immunoblotting. NAc synaptoneurosomes prepared from individual rats (10 μg protein/lane) were mixed 1:1 with 2× sample treatment buffer (catalog #161–0737, BIO-RAD) and analyzed by SDS-PAGE and immunoblotting. β-Tubulin was used as a loading control.