Heterogeneous Distribution of Mitochondria and Succinate Dehydrogenase Activity in Phrenic Motor Neurons

Neuromotor control of diaphragm muscle (DIAm) involves size-dependent recruitment of phrenic motor neurons (PhMNs). To accomplish breathing, only slow (type S) and fast, fatigue resistant (type FR) DIAm motor units are recruited. Thus, type s and FR DIAm motor units are very active in sustaining ventilation throughout life. Importantly, type S and FR DIAm motor units comprise small PhMNs that innervate type I and IIa DIAm fibers, respectively. More fatigable fast (type FF) motor units are very infrequently recruited for higher force, expulsive behaviors. Type FF DIAm motor units comprise large PhMNs that innervate type IIx/IIb DIAm fibers. Previously, we reported that compared to large PhMNs, small PhMNs have higher mitochondrial volume densities reflecting their more frequent activation. Using a quantitative histochemical technique to measure the maximum velocity of the succinate dehydrogenase reaction (SDH max ), we showed that type I and IIa DIAm fibers have higher oxidative capacity compared to type IIx/IIb fibers, which correlates with mitochondrial volume density and the more frequent activation of these muscle fibers. Accordingly, we hypothesize that the SDH max of small PhMNs is greater compared to large PhMNs. PhMNs in 6 month old, female and male Sprague Dawley rats were retrogradely identified by intrapleural injection of Alexa 647-conjugated cholera toxin B (CTB). Following retrograde CTB labeling, rats were perfused intracardially with a 0.1 M PBS, euthanized, and the C1–C6 segment of the spinal cord was dissected. Spinal cord sections were cut (40 mm) in longitudinal horizontal sections and mounted in 2-well Ibidi μ-slide plates. To measure SDH max , the sections were exposed to a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT; reaction indicator). As the SDH reaction proceeded, CTB-labeled PhMNs were imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion x60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD) was measured every 15 s over a 10-min period to measure the rate of accumulation of NBT diformazan (NBT dfz ; peak absorbance of 570 nm) in PhMNs. Using the Beer-Lambert equation, the change in OD was used to determine SDH max expressed as mmol fumarate/L cell/min. In separate experiments, following retrograde CTB labeling, mitochondria in PhMNs were labeled by transdural infusion of MitoTracker Red and imaged in 3D to determine mitochondrial volume density. Our results support a size-dependent heterogeneity in PhMN mitochondrial volume density and SDH max . We conclude that more active small PhMNs have a higher mitochondrial volume density and corresponding SDH max compared to large PhMNs. NIH grant AG44615 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.