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

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 (ΔΨm) 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 → ΔΨm 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.