Measurements of motoneuron somal volumes using laser confocal microscopy: Comparisons with shape-based stereological estimations

Previous studies on motoneuronal morphometry have assumed the geometry and orientation of neuronal soma in estimating somal volumes based on two-dimensional measurements. In this study, the optical sectioning property of the confocal microscope was used to make direct measurements of phrenic motoneuron somal volume. These measured volumes were compared to shape-based stereological estimates of volume. Phrenic motoneuron pools in adult rats were retrogradely labeled with fluorescent dye. Labeled motoneurons, in 150-/μm-thick tissue sections, were imaged using a Bio-Rad MRC 500 confocal microscope. Somal volumes were directly measured using ANALYZE, a comprehensive image processing software package. These volumes were compared to volume estimates based on five geometrical shapes previously used to study spinal motoneurons. In the adult phrenic motoneuron pool, overestimations of somal volume up to 300% were observed in cases where the Z-axis dimensions of the neurons were not simply related to the X and Y dimensions. None of the five geometrical shapes were found to be suitable for estimating either mean or individual somal volumes of the phrenic motoneuron pool. Confocal microscopy allowed accurate reconstruction along X, Y, and Z axes, and therefore provided a more direct method of measuring motoneuron somal volumes. We conclude that significant and inconsistent errors can be introduced by using shape-based stereological methods for estimating neuronal somal volumes.