Three-Dimensional Mapping of Retrograde Multi-Labeled Motor Neuron Columns in the Spinal Cord

The quantification and distribution characteristics of spinal motor neurons play important roles in the study of spinal cord and peripheral nerve injury and repair. In most research, the sole retrograde labeling of each nerve or muscle could not simultaneously obtain the distributions of different motor neuron subpopulations. Therefore, it did not allow mapping of spatial relationships of different motor neuron columns for disclosing the functional relationship of different nerve branches. Here, we combined the multiple retrograde labeling, optical clearing, and imaging for three-dimensional (3D) visualization of motor neurons of multiple brachial plexus branches. After screening fluorescent tracers by the labeling feasibility of motor neurons and fluorescence compatibility with optical clearing, we performed mapping and quantification of the motor neurons of ulnar, median, and radial nerves in the spinal cord, then disclosed the relative spatial distribution among different neuronal subpopulations. This work will provide valuable mapping data for the understanding of the functional relationships among brachial plexus branches, hopefully facilitating the study of regeneration of axons and remodeling of motor neurons in peripheral nerve repair.