A 3D atlas of sexually dimorphic lumbosacral motor neurons that control and integrate pelvic visceral and somatic functions in rats

The lumbosacral spinal motor system comprises somatic motor neurons (SMNs) that target striated muscle and visceral motor neurons (VMNs; autonomic preganglionic neurons) that target peripheral ganglia. The brain coordinates these pathways to mediate functions such as continence, voiding and sexual function by ensuring appropriate changes of organ state with striated muscle activity (e.g., sphincter opening, relevant postural changes). These spinal circuits are important therapeutic targets for bioelectronic medicine yet are poorly defined in comparison with limb SMNs. We aimed to define the structural features and relationships between SMNs and VMNs relevant to pelvic function in male and female rats, by building 3D maps of lumbosacral spinal cord. We achieved this by applying large-volume immunostaining (choline acetyltransferase), with tissue clearing and advanced whole mount microscopy (light sheet and ribbon scanning confocal microscopy). We identified VMNs specific to pelvic organ function by microinjecting cholera toxin (beta-subunit) into the major pelvic ganglia (MPG). These VMNS are primarily located in segments L1-L2 (dorsal commissural nucleus) and L6-S1 (intermediolateral nucleus). Unexpectedly, small groups of SMNs in the urethral rhabdosphincter, cremaster and levator ani motor pools also projected through the MPG. Motor neuron counts and analysis of dendritic fields detected sexual dimorphism in both SMNs and VMNs. Their colocation of dendritic bundles suggested a mechanism of coordinating activity. This study has provided the first multiscale 3D atlas of rat lumbosacral cord. This will be shared as a resource on an open science platform (sparc.science) to allow further exploration and modeling of network features and cellular morphology.