Clarification of the Innervation of the Bladder, External Urethral Sphincter and Clitoris: A Neuronal Tracing Study in Female Mongrel Hound Dogs

Abstract

Many studies examining the innervation of genitourinary structures focus on either afferent or efferent inputs, or on only one structure of the system. We aimed to clarify innervation of the bladder, external urethral sphincter (EUS) and clitoris. Retrograde dyes were injected into each end organ in female dogs. Spinal cord, mid-bladder, and spinal, caudal mesenteric, sympathetic trunk and pelvic plexus ganglia were examined for retrograde dye-labeled neurons. Neurons retrogradely labeled from the bladder were found primarily in L7-S2 spinal ganglia, spinal cord lateral zona intermedia at S1-S3 levels, caudal mesenteric ganglia, T11-L2 and L6-S2 sympathetic trunk ganglia, and pelvic plexus ganglia. The mid-bladder wall contained many intramural ganglia neurons labeled anterogradely from the pelvic nerve, and intramural ganglia retrogradely labeled from dye labeling sites surrounding ureteral orifices. Neurons retrogradely labeled from the clitoris were found only in L7 and S1 spinal ganglia, L7-S3 spinal cord lateral zona intermedia, and S1 sympathetic trunk ganglia, and caudal mesenteric ganglia. Neurons retrogradely labeled from the EUS were found in primarily at S1 and S2 spinal ganglia, spinal cord lamina IX at S1-S3, caudal mesenteric ganglia, and S1-S2 sympathetic trunk ganglia. Thus, direct inputs from the spinal cord to each end organ were identified, as well as multisynaptic circuits involving several ganglia, including intramural ganglia in the bladder wall. Knowledge of this complex circuitry of afferent and efferent inputs to genitourinary structures is necessary to understand and treat genitourinary dysfunction. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Keywords: caudal mesenteric ganglia; clitoris; detrusor muscle; external urethral sphincter; spinal cord; spinal ganglion; sympathetic trunk ganglia.

Figure 1

Cartoon of dye labeling sites. The location of dye injections in each end organ is shown for the (A) bladder (four sites surrounding the ureteral opening into the bladder), (B) external urethral sphincter (four sites) and (C) clitoris (two sites). Injection sites are indicated by a plus (+) sign. EUS = external urethral sphincter.

Figure 2

Spinal ganglia (SG; sensory) innervation of genitourinary structures. The mean number of retrogradely labeled cells in SG of T10 through S3 vertebral levels is shown for each end organ. (A) Mean number of labeled neurons/mm² from the bladder. (B) Mean number of labeled neurons/mm² from the clitoris. (C) Mean number of labeled neurons/mm² from the external urethral sphincter. (D) Fluorogold (FG) labeled cells from the bladder and True Blue (TB) labeled cells from the external urethral sphincter in a SG at S1. (E) Fluororuby and Nuclear Yellow labeled cells from the clitoris in SG at S1. (F) True Blue (TB) labeled cells from the external urethral sphincter in a SG at S1. FG = Fluorogold retrograde dye; L= lumbar; S= sacral; T= thoracic; TB = True Blue retrograde dye. Number (n) of dogs counted per end organ is indicated in panels A-C. Mean ± SEM shown.

Figure 3

Innervation of genitourinary structures directly from the spinal cord. Mean number of the retrogradely labeled neurons/mm² from the bladder (A), clitoris (B), and external urethral sphincter (C) in ventral horns of spinal cord levels indicated. Number (n) of dogs counted per end organ is indicated in each panel. T= thoracic; L= lumbar; S= sacral. Mean ± SEM shown. (D) A S1 ventral horn section stained with Nissl showing representative topographical mapping results of the location of neurons that were retrogradely labeled with Fluorogold (FG, green-yellow “O’s”), Nuclear Yellow (NY, large yellow and black “O’s”), or True Blue (TB, blue “O’s”). Each “O” surrounds a labeled neuron. Fluorogold and Nuclear Yellow labeled neurons were located in lamina VII, a zone typically associated with autonomic function. In contrast, True Blue labeled neurons were located in medial and lateral regions of lamina IX, a zone typically associated with somatic motor function. (E) Fluorogold and Nuclear Yellow (NY) labeled neurons in an S2 ventral horn section. (F) True Blue labeled neurons in an S1 ventral horn section.

Figure 4

Caudal Mesenteric Ganglia (sympathetic) innervation of genitourinary structures. Mean number of retrogradely labeled neurons/mm² from the bladder, external urethral sphincter (EUS) and clitoris. Six dogs were counted per end organ. Mean ± SEM shown.

Figure 5

Sympathetic trunk ganglia innervation of genitourinary structures. Mean number of retrogradely labeled neurons/mm² from (A) the bladder, (B) the external urethral sphincter, and (C) the clitoris in sympathetic trunk ganglia located at the vertebral levels shown. Number (n) of dogs counted per end organ is indicated in each panel. L= lumbar; S= sacral; T= thoracic. Mean ± SEM shown.

Figure 6

Innervation of genitourinary structures from pelvic plexus ganglia. Mesenteries adjacent to the bladder containing pelvic plexus ganglia were collected, fixed, and flat mounted by placing into petri dishes. The number of ganglia and retrogradely labeled cells per ganglia were counted, without sectioning, using an inverted microscope. (A) Mean number (± SEM) of pelvic plexus ganglia per dog. (B) Mean number of labeled neurons/mm² in each pelvic plexus ganglia from the bladder, external urethral sphincter and clitoris. Five dogs were counted per end organ. (C) Flat mounted mesenteries in a petri dish visualized using both bright field and UV fluorescent lights. Fluorogold labeled cells from the bladder are visible as bright blue dots. (D) A higher power as same region indicated in panel B by the arrow; visualized using UV fluorescence light only. Several groupings of Fluorogold labeled cells are visible. Inset shows higher power of labeled cells indicated with arrow. (E) A different mesenteric region showing Fluorogold labeled cells from the bladder at even higher power so that nuclei are visible.

Figure 7

Intramural ganglia in the bladder detrusor muscle. (A) Hematoxylin and eosin (H&E) stained intramural ganglion in the detrusor muscle. m = nearby detrusor smooth muscle fiber. Inset shows a higher power image of the neuron indicated with an arrow. (B) Neurofilament-200 (NF) immunostained (DAB brown staining) intramural ganglion (g) and associated nerve fibers (n). m = nearby detrusor smooth muscle fibers. (C) Fluorogold (FG, cells containing pale blue-gold yellow dots) stained neurons in an intramural ganglion (arrows). DAPI (a bright blue nuclear stain) was used as a counterstain. Nuclei of Fluorogold labeled neurons and glial cells present in the ganglia. (D) Same cells indicated with arrows in panel C were co-labeled with PGP 9.5 (green staining; a pan neuronal marker). Cells indicated with asterisks in panels C and D are PGP 9.5+ neurons that are not double-labeled with Fluorogold. (E) Postmortem anterograde DiI labeling in an anterior vesicle branch of the pelvic nerve (n), shown entering the bladder. (F) An intramural ganglion (g) and associated nerve fibers (n) labeled with DiI.

Figure 8

Diagram of typical sensory, sympathetic, parasympathetic and somatic motor innervation of the bladder and external urethral sphincter (EUS) in dogs. Sensory input (dotted black arrow) project to SG primarily at levels L6-S2 of the spinal cord from the bladder, levels L7-S1 from the clitoris, and levels S1-S2 from the external urethral sphincter. Preganglionic sympathetic axons (dashed red arrow) to pelvic viscera originate from preganglionic neurons in lumbar spinal cord segments and contribute to: 1) sympathetic trunk ganglia coursing the length of the vertebral column; 2) lumbar splanchnic nerves to the caudal (inferior) mesenteric ganglia (many via the hypogastric nerve). Postganglionic sympathetic axons to the bladder originate mostly from L7-S2 sympathetic ganglia and caudal mesenteric ganglion. Note that the sensory and sympathetic axons hitchhike on other nerve bundles and arteries. Parasympathetic innervation (solid blue arrow) of the bladder originates primarily from S1-S3 and often synapse on pelvic plexus ganglia adjacent to the bladder. The external urethral sphincter is innervated by somatic motor inputs (solid green arrow) originating from the sacral cord. The function of direct inputs from the lower thoracic and upper lumbar spinal cord (solid grey arrow) to the bladder is currently unknown. EUS = external urethral sphincter; CMG = caudal mesenteric ganglion (referred to as the inferior mesenteric ganglion in human anatomy); PPG = Pelvic Plexus Ganglia (multiple) located in mesenteries of bladder.