Quantifying mitochondrial volume density in phrenic motor neurons

Abstract

BackgroundPrevious assessments of mitochondrial volume density within motor neurons used electron microscopy (EM) to image mitochondria. However, adequate identification and sampling of motor neurons within a particular motor neuron pool is largely precluded using EM. Here, we present an alternative method for determining mitochondrial volume density in identified motor neurons within the phrenic motor neuron (PhMN) pool, with greatly increased sampling. New methodThis novel method for assessing mitochondrial volume density in PhMNs uses a combination of intrapleural injection of Alexa 488-conjugated cholera toxin B (CTB) to retrogradely label PhMNs, followed by intrathecal application of MitoTracker Red to label mitochondria. This technique was validated by comparison to 3D EM determination of mitochondrial volume density as a “gold standard”. ResultsA mean mitochondrial volume density of ∼11 % was observed across PhMNs using the new MitoTracker Red method. This compared favourably with mitochondrial volume density (∼11 %) measurements using EM. Comparison with existing methodThe range, mean and variance of mitochondrial volume density estimates in PhMNs were not different between EM and fluorescent imaging techniques. ConclusionsFluorescent imaging may be used to estimate mitochondrial volume density in a large sample of motor neurons, with results similar to EM, although EM did distinguish finer mitochondrion morphology compared to MitoTracker fluorescence. Compared to EM methods, the assessment of a larger sample size and unambiguous identification of motor neurons belonging to a specific motor neuron pool represent major advantages over previous methods.