Quantifying mitochondrial volume density in phrenic motor neurons

Area of Science:

• Neuroscience
• Cell Biology
• Biophysics

Background:

• Traditional electron microscopy (EM) for assessing mitochondrial volume density in motor neurons has limitations in identifying and sampling specific motor neuron pools.
• Previous methods using EM are insufficient for comprehensive analysis within a defined motor neuron pool.

Purpose of the Study:

• To introduce and validate a novel fluorescent imaging technique for determining mitochondrial volume density in identified phrenic motor neurons (PhMNs).
• To overcome the sampling limitations of EM for motor neuron pool analysis.

Main Methods:

• The method involves retrograde labeling of PhMNs using Alexa 488-conjugated cholera toxin B (CTB) via intrapleural injection.
• Mitochondria within labeled PhMNs are subsequently stained with MitoTracker Red via intrathecal application.
• The technique's accuracy was validated against 3D EM, considered the gold standard.

Main Results:

• The novel fluorescent imaging method yielded a mean mitochondrial volume density of approximately 11% in PhMNs.
• These results closely matched the mitochondrial volume density measurements obtained using EM.
• No significant differences were found in the range, mean, or variance of mitochondrial volume density estimates between the EM and fluorescent imaging techniques.

Conclusions:

• Fluorescent imaging offers a viable alternative to EM for estimating mitochondrial volume density in large samples of motor neurons, providing comparable results.
• This method allows for unambiguous identification of specific motor neuron pools and significantly increases sample size compared to EM.
• While EM provides finer morphological detail, the new fluorescent technique offers major advantages in sampling and identification for motor neuron pool studies.