Measurement of mitochondrial membrane potential

Mitochondrial membrane potential was measured based on a previous protocol (44), and the fluorescent dye tetramethylrhodamine methyl ester (TMRM, Invitrogen) was used because it accumulates in mitochondria based on Δψ_m. Neurons were plated on 35 mm glass-bottomed dishes. They were washed three times with 5 mM K⁺, 2 mM Ca²⁺ Tyrodes solution, then incubated with 10 nM TMRM in 2 ml Tyrodes solution for 45 min at room temperature in the dark. Live-cell imaging was performed using a Zeiss Axiovert 200M microscope equipped with an incubation chamber, using an EC Plan-Neofluar 40×/1.30 Oil DIC objective. Cells were kept at 37°C with 5% CO₂ while imaging. Microscope settings were optimized using control cells, and these same settings were used for all other groups. Randomly selected fields were imaged every 20 s for a total of 600 s. The mitochondrial uncoupler FCCP was added to the media after 300 s to a final concentration of 1 µM. Time series were analyzed in MetaMorph, and at least 20 regions of interest (ROIs) were traced around mitochondrial structures for each cell, along with adjacent background regions. The pixel intensity for each region was determined, followed by background subtraction. Changes in fluorescence were calculated with the formula ΔF = (F − F₀)/F₀ × 100, where F is the fluorescence intensity at any time point, and F₀ is the initial fluorescence.