Synaptosomal Fraction Isolation Protocol

The synaptosomal fractions were obtained as described previously^{28 (link)}. Briefly, the tissue was homogenized using a Dounce homogenizer in 0.32 M Sucrose, 10 mM Tris–HCl (pH 7.4) buffer (buffer A) containing complete protease and phosphatase inhibitor cocktails (Sigma). After homogenization, the crude synaptosomal fraction (synaptosomes plus mitochondria) was isolated by two sequential centrifugations (1,500×g, 10 min followed by 12,500×g, 20 min; at 4 °C). The crude synaptosomes were resuspended in 13% (final concentration) Ficoll 400 (in buffer A) and layered on the bottom of a discontinuous gradient, composed by buffer A and 7% Ficoll (in buffer A). The gradients were centrifuged at 100,000×g (45 min at 4 °C) and the synaptosomes were isolated at the 7.5–13% interface. After washing (twice with buffer A), the protein content of the synaptosomal fractions was quantified by Lowry.