Mouse brain tissue was homogenized in a buffer containing 0.32 M sucrose in PBS, supplemented with protease/phosphatase inhibitors.

The homogenate was centrifuged at 1,000 × g for 10 min at 4 °C to

remove nuclei and cellular debris (P1). The supernatant (S1) was centrifuged at

15,000 g g for 15min at 4 °C to yield a crude synaptosomal pellet (P2).

This pellet was hypo-osmotically lysed in water containing protease inhibitors for 5 min at 4

°C and passed through both a 22- and 27½-gauge needle (10 times each).

This suspension was centrifuged at 23,000 × g for 22 min at 4 °C, and the resulting supernatant LS1(S3) was centrifuged again at 174,900 × g for 2 hours at 4 °C in a STi32 Beckman rotor. The final pellet (LP2 or P4) containing an enriched fraction of synaptic vesicles was used for subsequent

experiments.