Measuring Mitochondrial Respiration in DRG Neurons

Oxygen consumption rate (OCR) was assessed in intact lumbar DRG sensory neurons using the XF96 extracellular flux analyzer (Seahorse Biosciences, North Billerica, MA). Lumbar sensory neurons were dissected from two to three mice and pooled together, and the neurons were isolated as previously described.^{21 (link)} The cells were maintained overnight in Ham’s F10 medium (6.1 mM glucose) containing 50 ng/mL nerve growth factor, 1 ng/mL neurotrophin 3 and N2 supplement without insulin (Invitrogen, Carlsbad, CA). The following day, the medium was changed to unbuffered DMEM supplemented with 1 mM sodium pyruvate and 5.5 mM d-glucose, and the cells were incubated at 37 °C for 1 h before being introduced to the XF96 analyzer. The initial readings provide a measure of the basal OCR before the addition of respiratory chain poisons.^{61 (link)} The addition of oligomycin (1 µg/mL), an ATP synthase inhibitor, leads to a decrease in OCR which represents the portion of basal OCR that is coupled to ATP synthesis. The residual OCR that persists after addition of oligomycin is from uncoupled respiration (proton leak). Next, maximal respiratory capacity (MRC) was assessed by adding 1 µM FCCP, a protonophore that dissipates the proton gradient across the inner mitochondrial membrane. Nonmitochondrial respiration was then assessed by co-injection of 1 µM rotenone + 1 µM antimycin A. After the respiratory measures, the cells were harvested, and OCR values were normalized to the total protein content of each well. Maximal respiratory capacity (MRC) and spare respiratory capacity (SRC) were quantified as described previously.^{61 (link),62 (link)}