Synaptosome isolation

Synaptosomes were generated from forebrains, hippocampi, or cerebella of 6- to 8-week-old wild-type and vGLUT1^VENUS knock-in mice as described previously (27, 28, 30). Our synaptosome preparation was chosen and adapted from our previously published protocol (27–29) to favor the isolation of synaptosomes with presynaptic compartments with closed membrane bilayers and postsynaptic compartments with open membrane bilayers. Briefly, the cerebellum, the forebrain, or both hippocampi from a single mouse were homogenized in 2 ml of ice-cold homogenization buffer [0.32 M sucrose, 4 mM HEPES (pH 7.4), EGTA-free protease inhibitor cocktail (Calbiochem, 1:1000), and RNasin (Promega, 1:1000)] by using a 2-ml glass-Teflon homogenizer with 12 gentle strokes. The homogenizer was then rinsed with an additional 3 ml of homogenization buffer, and the combined 5 ml of homogenate was centrifuged at 1000 × g for 8 min at 4°C. The supernatant was centrifuged again at 12,500 × g for 15 min at 4°C. The synaptosome-enriched pellet was then resuspended in 1 ml of homogenization buffer. This fraction was finally layered on top of a two-step sucrose density gradient (5 ml of 1.2 M sucrose and 5 ml of 0.8 M sucrose, 4 mM HEPES, and EGTA-free protease inhibitor cocktail, as described above). The gradient was centrifuged at 50,000 × g for 70 min at 4°C. Synaptosomes were recovered through the tube wall, at the interface of 0.8 and 1.2 M sucrose, by using a syringe to minimize contamination with lighter fractions enriched in myelin. The resulting fraction is referred to as presorted (sucrose) synaptosomes (or S-synaptosomes) as opposed to vGLUT1⁺ sorted synaptosomes (or FASS-synaptosomes).