Mitochondrial respiration measurements

To assess mitochondrial respiration, oxygen consumption rates (OCR) were measured in hippocampal neurons using a Seahorse XFp Analyzer with Cell Mito Stress Kit (Agilent Technologies) in accordance with the manufacturer’s instructions. Medium composition and inhibitor concentrations were determined following a published guideline for assessing mitochondrial function in primary neurons (Connolly et al., 2018). Briefly, dissociated embryonic hippocampal neurons were plated at a density of 100,000 cells/well in Seahorse XFp mini-plates pre-coated with poly-D-lysine and laminin, and cultured for 15 days before treatment. On the day of the assay (DIV15-DIV18), cells were washed and equilibrated in experimental buffer consisting of low-buffered bicarbonate-free XF DMEM medium (pH 7.4) supplemented with 10 mM glucose and 2 mM sodium pyruvate for 1 h at 37°C with no CO₂. Baseline rates were measured at 37°C before the sequential injection of oligomycin (6 μM), FCCP (3 μM), and rotenone/antimycin A cocktail (0.5 μM). Basal OCR levels were determined by subtracting the non-mitochondrial respiration rate from the last respiration rate measured before the oligomycin injection. ATP production was measured by subtracting the proton leak rate from the basal respiration rate. Maximum respiration was calculated by subtracting the non-mitochondrial respiration rate from the maximum rate measurement after FCCP injection. All measurements were normalized to DNA content per well using the CyQUANT assay (Thermo Fisher Scientific).