Abstract: To perform various functions in the body, skeletal muscle is controlled and coordinated as a whole by nerves. However, there has been little research into whether the nerve control characteristics of different muscles are different, and the importance of these potential differences. In the present study, we used a three-dimensional imaging of solvent-cleared organ-compatible multi-tracer technique to explore the spatial distribution patterns of sensory and sympathetic neurons that innervate limb muscles. We integrated transcriptome sequencing datasets from mouse limb muscles in public databases and performed correlation analysis with neuronal spatial distribution data to reveal the unique effects of different types of neurons on muscle functional pathways. In terms of spatial distribution patterns, sympathetic neurons exhibited a more concentrated distribution than sensory and motor neurons. In addition, the neuronal innervation of limb muscles exhibited four different characteristics: sympathetic neuron-rich muscle, sensory neuron-rich muscle, neuron-sparse muscle, and motor neuron-rich muscle. Sensory neuron density was mainly associated with muscle contractile structure and cell pH, whereas sympathetic neuron density was associated with protein kinase activity, muscle vasculature, muscle calcium-dependent protein kinase activity, lipid transport, and vesicle release. Motor neuron density was mainly associated with protein kinase activity, cell adhesion, oxidoreductase activity, and exocytosis. These findings may contribute to a deeper understanding of how nerves cooperate to endow muscles with diverse physiological functions, thereby providing new insights and experimental evidence for the treatment of various neuromuscular diseases.

Key words: 3D imaging, dorsal root ganglia, motor neuron, retrograde tracing, skeletal muscle, sympathetic ganglion, transcriptome