SPARC: A Multiscale Analysis of the Structure of the Rat Lower Urinary Tract (LUT) and Connectivity of Sensory and Autonomic Nerve Terminals

Abstract

Storage and voiding of urine are normally precisely controlled to occur only in appropriate physiological and behavioral contexts. Neural control is provided by the projecting terminals of extrinsic sensory, autonomic and somatomotor neurons, interfacing with a spinal cord‐brain network that coordinates lower urinary tract activity with patterned activity of other pelvic organs (e.g. colon), somatic structures (e.g. pelvic floor, hind‐limbs). Multiple anatomical and functional classes of peripheral nerves project to the LUT but it has not yet been determined how the microscopic connectivity and circuit organisation of this neural interface control basic organ functions. Our aim was to address this question by developing a workflow for multiscale peripheral connectome analysis that could 1) segment the LUT into functional anatomical units, 2) understand the organisational principles by which these units are targeted by the projecting terminals of different functional classes of sensory, autonomic motor and somatomotor neurons, and 3) produce analysis and 3D digital visualisations of this connectivity at macroscopic (whole organ), mesoscopic (tissue compartments) and microscopic (single axon connectivity) scales. The studies were performed using adult male and female Sprague‐Dawley rats. We first used micro‐CT scanning to visualise the macroscopic organisation of the LUT. This included entry points of the ureters, bladder dome, bladder neck, urethra and external urinary sphincter (i.e., striated muscle rhabdosphincter). The resolution of this imaging also proved sufficient for first‐round segmentation of mesoscopic tissue compartments within the walls of the organ. These data enabled iterative development of a digital mesh‐based scaffold as a platform to support further visualisations and digital mapping at higher resolutions. To do this LUT organs were removed, cleared using iDISCO, and immunostained using tissue specific markers (e.g. smooth muscle actin; Acta1 for striated muscle). The intact organs were then imaged with light sheet microscopy or flattened whole mounts with widefield or confocal microscopy. This resulted in visualisations of the complex organisation of the smooth muscle bands within the connective tissue matrix of the bladder dome and neck; the trigone defined by the entry points of the ureters; and structural associations of urethral compartments with the surrounding rhabdosphincter and vasculature. To subclassify projecting nerve terminals and visualise targeting of single axons with cellular targets we extended our analyses by 1) using sparse labelling with reporter adeno‐associated viruses (AAVs) as neural tracers, and 2) using ribbon‐scanning confocal microscopy for large volume imaging of nerve terminals at submicron resolution in some samples. We have determined this integrated workflow combining multiple imaging methodologies with digital mapping can support multiscale analysis required to understand the functional organisation of the neural interface that encodes LUT sensation and controls LUT function.Support or Funding InformationNIH SPARC 3OT2OD023872‐01S4.