Synaptosome Extraction and Characterization

Synaptosomes were extracted using Syn-PER Reagent as per manufacturer instructions with some modifications (Thermo Scientific, USA)^{48 (link),49 (link),60 (link)}. Briefly, 50 mg of brain tissue was used from each sample for synaptosome extraction in 1 ml of Syn-PER Reagent. Tissues were homogenized slowly by Dounce glass homogenization on ice with ~10 slow strokes. The resulting tissue homogenates were transferred to a centrifuge tube. Samples were centrifuged at 1400×g for 10 minutes at 4 °C to remove the leftover tissue debris. After centrifugation, the supernatant was transferred to a new tube. Again, supernatant (homogenate) was centrifuged at high-speed 15,000 × g for 20 min at 4 °C. The supernatant was removed as a cytosolic fraction and synaptosomes recovered in the pellet form. Both the cytosolic fraction and synaptosome pellet were processed for RNA and protein extraction. The synaptosome pellet was also processed for transmission electron microscopic (TEM) analysis.