Base media

To study whether cisplatin treatment resulted in altered mitochondrial morphology at the time of the behavioral analyses, we prepared synaptosomes of the brains of mice at 7–9 days after the last dose of cisplatin. Synaptosomes were isolated according to Kamat et al. [29 (link)]. Briefly, after perfusion with PBS, brains were dissected and homogenized (10% w/v) into a 0.32 M sucrose 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (145 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, 5 mM HEPES, pH 7.4) using a glass Dounce homogenizer with ~10 up-and-down strokes. The brain lysate was centrifuged at 4°C for 10 min at 1000× g. The supernatant was extracted and diluted 1:1 with 1.3 M sucrose HEPES buffer (final concentration of 0.8 M sucrose) and centrifuged at 4°C for 30 min at 20,000× g. The pellet containing isolated synaptosomes was resuspended in base media (Seahorse Biosciences/Agilent Technologies, Santa Clara, CA) supplemented with 11 mM glucose, 2 mM glutamine, and 1 mM pyruvate for oxygen-consumption analysis or 2% glutaraldehyde plus 2% PFA in PBS for transmission electron microscopy.