Action potential-induced changes in O₂ consumption, NADH regeneration, and mitochondrial membrane potential in adult...

Studies of central neurons suggest that stimulation activates Ca²⁺-dependent processes that increase NADH regeneration and O₂ consumption. This study asked whether similar processes dominate respiratory responses in spinal motor neurons. Lumbar ventral horn segments from adult mice were perfused in a flow-through chamber. Motor neurons were stimulated with trains of action potentials (5-80 Hz for 20-40 s) conducted antidromically from ventral roots. Perfusion solutions contained antagonists of ionotropic cholinergic and glutaminergic receptors to inhibit synaptic transmission. The stimulation-induced increase in O₂ consumption, calculated from changes in the phosphorescence/fluorescence of porphyrin compounds, was inhibited reversibly by procaine and low [K⁺] solutions, and blocked by inhibitors of mitochondrial respiration (rotenone, oligomycin). These findings suggest that the increase in O₂ consumption was due mainly to activation of ATPase pumps to counteract the changes in transmembrane ion gradients produced by repetitive action potential discharge. In glucose, pyruvate, or lactate (1-10 mM), peak O₂ consumption and NADH regeneration increased with action potential workload. For all three tested substrates, a fraction of O₂ consumption (median 12%-40%) was Ca²⁺-dependent; NADH regeneration showed significant Ca²⁺ dependence only in low [lactate] (2.5 mM). Stimulation produced a depolarizing shift in the mitochondrial membrane potential (ΔΨ_m) that increased with workload. The relationship between O₂ consumption and NADH regeneration was flexible, in a manner suggesting that resting NADH is sufficient for the initial respiratory response to stimulation. A scenario consistent with most respiration in glucose is action potential workload →increased pump activity →↑ADP/ATP ratio → ↑ATP synthase activity → ΔΨ_m depolarization→↑O₂ consumption.NEW & NOTEWORTHY In a novel preparation of adult mouse motor neurons stimulated by back-propagated action potentials, the relationship between O₂ consumption and NADH regeneration was flexible. Data suggest a resting reservoir of NADH sufficient to support the response to short-term workloads. In glucose, most O₂ consumption and NADH regeneration were independent of extracellular [Ca²⁺]. The major driver of the respiratory response to stimulation was an increased ADP/ATP ratio, activating ATP synthase, which uses the mitochondrial proton gradient.