SPARC: Building a Three‐Dimensional Atlas of Spinal Cord Circuits Regulating Lower Urinary Tract Function

Abstract

Lumbar and sacral spinal cord circuits are critical for pelvic visceral regulation, as well as being engaged in pelvic pain states. Our study has focused on mapping two components of these circuits, the central projections of primary sensory neurons and the preganglionic neurons that drive motor activity via pelvic ganglia. These sensory pathways form a minority of lumbosacral dorsal root ganglion neurons. Likewise, although the parasympathetic preganglionic neurons are easily identified (aggregated in the sacral intermediolateral nucleus), the sympathetic preganglionic neurons comprise a minority of the thoracolumbar preganglionic neurons and are largely located outside of the intermediolateral nucleus. Our specific objectives were to map in adult male and female SD rats: (i) the terminal fields of primary sensory pathways that innervate distinct regions of the lower urinary tract; (ii) sympathetic and parasympathetic preganglionic neurons that innervate pelvic ganglion neurons, many of which regulate lower urinary tract tissues. To facilitate this, we have developed a spinal cord reference atlas, utilizing large volume immunostaining and clearing methods (iDISCO) to visualize primary neural classes throughout the entire lumbosacral cord. Region‐specific sensory neurons were identified by micro‐injection of CTB (B subunit of cholera toxin) into the bladder body, bladder trigone or urethra. In a separate group of animals, pelvic ganglion‐projecting preganglionic neurons were identified by microinjecting CTB bilaterally into the major pelvic ganglion (MPG). Four days later, spinal cord tissue was removed for immunohistochemical processing. We then combined several imaging approaches to reveal both mesoscopic and microscopic anatomical features of these pathways. Sensory terminals were mapped by constructing three‐dimensional representations, aligning alternate cryosections (TissueMaker, MicroBrightField) and relating CTB‐labelled terminals to specific chemoarchitectural features including spinal laminae and preganglionic neurons. Several molecular subclasses of sacral CTB‐labelled preganglionic neurons were mapped using the same strategy, while in other studies, the MPG‐projecting preganglionic neurons were mapped across the entire lumbosacral spinal cord, using immunohistochemistry of intact specimens and iDISCO clearing. Maps were constructed using 3D datasets obtained by light sheet microscopy or, for higher resolution analyses of neuronal connectivity and dendritic structure, ribbon scanning confocal microscopy. In conclusion, our tracing studies provide new insights into the complexity of sensory projections from defined pelvic visceral structures, and the location and structural properties of specific classes of pelvic viscera‐regulating preganglionic neurons. Our new spinal cord reference dataset provides a rich resource for mapping neural features following experimental intervention, for studies across the autonomic nervous system.Support or Funding InformationNIH SPARC 3OT2OD023872‐01S4