Abstract: Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneurons. However, current research on the spatial location of the spinal motoneurons innervating different muscles is limited. In this study, we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs, which were innervated by the obturator nerve, femoral nerve, inferior gluteal nerve, deep peroneal nerve, and tibial nerve. Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared organs (3DISCO) and 3-D imaging technology based on lightsheet fluorescence microscopy (LSFM). Additionally, we propose the hypothesis that “messenger zones” exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups. We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons. Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse. Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles, but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.

Key words: 3-D imaging, motoneurons, multiple retrograde tracing, muscle coordination, skeletal muscle, spatial distribution, optical tissue clearing