dissection-pelvic-autonomic-ganglia-associated-nerves-male-female

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

University of Melbourne

The nerve growth factor (NGF) precursor, proNGF, is implicated in various neuropathological states. ProNGF signals apoptosis by forming a complex with the receptors p75 and sortilin, however, it can also induce neurite growth, proposed to be mediated by the receptor of mature NGF, tyrosine kinase receptor A (TrkA). The way in which these dual effects occur in adult neurons is unclear. We investigated the neurotrophic effects of proNGF on peptidergic sensory neurons isolated from adult mouse dorsal root ganglia and found that proNGF stimulated neurite extension and branching, requiring p75, sortilin and TrkA. Neurite growth rarely occurred in sortilin-expressing neurons but was commonly observed in TrkA-positive, sortilin-negative neurons that associated closely with sortilin-positive glia. ProNGF was unable to induce local trophic effects at growth cones where sortilin-positive glia was absent. We propose that in adult sensory neurons the neurotrophic response to proNGF is mediated by NGF and TrkA, and that peri-somatic glia may participate in sortilin- and p-75 dependent cleavage of proNGF. The potential ability of local glial cells to provide a targeted supply of NGF may provide an important way to promote trophic (rather than apoptotic) outcomes under conditions where regeneration or sprouting is required.

Neurotrophic actions initiated by proNGF in adult sensory neurons may require peri-somatic glia to drive local cleavage to NGF.

Recent evidence suggests that the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli and signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder. This led us to establish a method of co-culturing dorsal root ganglion sensory neurons and bladder urothelial cells.

Co-cultures provide a new tool to probe communication between adult sensory neurons and urothelium.

Innervation of developing and adult organs.

Bladder sensation is mediated by lumbosacral dorsal root ganglion neurons and is essential for normal voiding and nociception. Numerous electrophysiological, structural, and molecular changes occur in these neurons following inflammation. Defining which neurons undergo these changes is critical for understanding the mechanism underlying bladder pain and dysfunction. Our first aim was to define the chemical classes of bladder sensory neurons that express receptors for the endogenous modulators of nociceptor sensitivity, glial cell line-derived neurotrophic factor (GDNF), the related neurotrophic factor, artemin, and estrogens. Bladder sensory neurons of adult female Sprague-Dawley rats were identified with retrograde tracer. Diverse groups of neurons express these receptors, and some neurons express receptors for both neurotrophic factors and estrogens. Lumbar and sacral sensory neurons showed some distinct differences in their expression profile. We also distinguished the chemical profile of myelinated and unmyelinated bladder sensory neurons. Our second aim was to identify bladder sensory neurons likely to be undergoing structural remodeling during inflammation. Following systemic administration of cyclophosphamide (CYP), its renal metabolite acrolein causes transient urothelial loss, exposing local afferent terminals to a toxic environment. CYP induced expression of the injury-related immediate-early gene product, activating transcription factor-3 (ATF-3), in a small population of sacral nitrergic bladder sensory neurons. In conclusion, we have defined the bladder sensory neurons that express receptors for GDNF, artemin and estrogens. Our study has also identified a sub-population of sacral sensory neurons that are likely to be undergoing structural remodeling during acute inflammation of the bladder. Together these results contribute to increased understanding of the neurons that are known to be involved in pain modulation and hyperreflexia during inflammation. 

Characterization of bladder sensory neurons in the context of myelination, receptors for pain modulators, and acute responses to bladder inflammation.

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family that has been strongly implicated in development and regeneration of autonomic nerves and modulation of nociception. Whereas other members of this family (GDNF and neurturin) primarily target parasympathetic and nonpeptidergic sensory neurons, the artemin receptor (GFRα3) is expressed by sympathetic and peptidergic sensory neurons that are also the primary sites of action of nerve growth factor, a powerful modulator of bladder nerves. Many bladder sensory neurons express GFRα3 but it is not known if they represent a specific functional subclass. Therefore, our initial aim was to map the distribution of GFRα3-immunoreactive (-IR) axons in the female rat bladder, using cryostat sections and whole wall thickness preparations. We found that GFRα3-IR axons innervated the detrusor, vasculature, and urothelium, but only part of this innervation was sensory. Many noradrenergic sympathetic axons innervating the vasculature were GFRα3-IR, but the noradrenergic innervation of the detrusor was GFRα3-negative. We also identified a prominent source of nonneuronal GFRα3-IR that is likely to be glial. Further characterization of bladder nerves revealed specific structural features of chemically distinct classes of axon terminals, and a major autonomic source of axons labeled with neurofilament-200, which is commonly used to identify myelinated sensory axons within organs. Intramural neurons were also characterized and quantified. Together, these studies reveal a diverse range of potential targets by which artemin could influence bladder function, nerve regeneration, and pain, and provide a strong microanatomical framework for understanding bladder physiology and pathophysiology.

Characterization of axons expressing the artemin receptor in the female rat urinary bladder: a comparison with other major neuronal populations.

Following peripheral nerve injury, restoration of function may occur via the regeneration of injured axons or compensatory sprouting of spared axons. Injury to visceral nerves that control urogenital organs is a common consequence of pelvic surgery, however their capacity to reinnervate organs is poorly understood. To determine if and how sensory and motor connections to the bladder are re-established, a novel surgical model of visceral nerve injury was performed unilaterally in adult male Wistar rats. Bladder-projecting motor and sensory neurons in pelvic ganglia and lumbosacral dorsal root ganglia, respectively, were identified and characterised by retrograde tracing and immunofluorescence. Application of tracers ipsi- and contralateral to injury distinguished the projection pathways of new connections in the bladder. In naive animals, the majority of sensory and motor neurons project ipsilaterally to the bladder, while ~20 % project contralaterally and ~5 % bilaterally. Injured axons of motor neurons were unable to regenerate by 4weeks after transection. In contrast, by this time many injured sensory neurons regrew axons to reform a substantial plexus within the detrusor and suburothelial tissues. These regeneration responses were also indicated by upregulation of activating transcription factor-3 (ATF-3), which was sustained in motor neurons but transient in sensory bladder-projecting neurons. Axotomy had little or no effect on the survival of bladder-projecting sensory and motor neurons. We also found evidence that uninjured motor and sensory neurons develop additional projections to the denervated bladder tissue and return connectivity, likely by undergoing compensatory growth. In conclusion, our results show that visceral sensory and motor neurons have a different capacity to regenerate axons following axotomy, however in both components of the circuit uninjured bladder neurons spontaneously grow new axon collaterals to replace the lost terminal field within the organ. For a full functional recovery, understanding the environmental and cellular mechanisms that reduce the ability of pelvic ganglion cells to undergo axonal regeneration is needed. 

Regeneration of sensory but not motor axons following visceral nerve injury.

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect. 

Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin.

GDNF (glial cell line-derived neurotrophic factor), neurturin and artemin use their co-receptors (GFRα1, GFRα2 and GFRα3, respectively) and the tyrosine kinase Ret for downstream signaling. In rodent dorsal root ganglia (DRG) most of the unmyelinated and some myelinated sensory afferents express at least one GFRα. The adult function of these receptors is not completely elucidated but their activity after peripheral nerve injury can facilitate peripheral and central axonal regeneration, recovery of sensation, and sensory hypersensitivity that contributes to pain. Our previous immunohistochemical studies of spinal cord and sciatic nerve injuries in adult rodents have identified characteristic changes in GFRα1, GFRα2 or GFRα3 in central spinal cord axons of sensory neurons located in DRG. Here we extend and contrast this analysis by studying injuries of the pelvic and hypogastric nerves that contain the majority of sensory axons projecting to the pelvic viscera (e.g., bladder and lower bowel). At 7 d, we detected some effects of pelvic but not hypogastric nerve transection on the ipsilateral spinal cord. In sacral (L6-S1) cord ipsilateral to nerve injury, GFRα1-immunoreactivity (IR) was increased in medial dorsal horn and CGRP-IR was decreased in lateral dorsal horn. Pelvic nerve injury also upregulated GFRα1- and GFRα3-IR terminals and GFRα1-IR neuronal cell bodies in the sacral parasympathetic nucleus that provides the spinal parasympathetic preganglionic output to the pelvic nerve. This evidence suggests peripheral axotomy has different effects on somatic and visceral sensory input to the spinal cord, and identifies sensory-autonomic interactions as a possible site of post-injury regulation. 

Peripheral injury of pelvic visceral sensory nerves alters GFRα (GDNF family receptor alpha) localization in sensory and autonomic pathways of the sacral spinal cord.

Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external genitalia and lower reproductive structures. Representative illustrations, detailed text descriptions and molecular markers that selectively label muscle, nerves/ganglia and epithelia of the lower urogenital system are also presented. The revised ontology will be an important tool for researchers studying urogenital development/malformation in mouse models and will improve our capacity to appropriately interpret these with respect to the human situation. 

An illustrated anatomical ontology of the developing mouse lower urogenital tract.

The development of organs occurs in parallel with the formation of their nerve supply. The innervation of pelvic organs (lower urinary tract, hindgut, and sexual organs) is complex and we know remarkably little about the mechanisms that form these neural pathways. The goal of this short review is to use the urinary bladder as an example to stimulate interest in this question. The bladder requires a healthy mature nervous system to store urine and release it at behaviorally appropriate times. Understanding the mechanisms underlying the construction of these neural circuits is not only relevant to defining the basis of developmental problems but may also suggest strategies to restore connectivity and function following injury or disease in adults. The bladder nerve supply comprises multiple classes of sensory, and parasympathetic or sympathetic autonomic effector (motor) neurons. First, we define the developmental endpoint by describing this circuitry in adult rodents. Next we discuss the innervation of the developing bladder, identifying challenges posed by this area of research. Last we provide examples of genetically modified mice with bladder dysfunction and suggest potential neural contributors to this state. 

Developing a functional urinary bladder: a neuronal context.

Renaming all spinal autonomic outflows as sympathetic is a mistake.

Controversy exists in the literature regarding the presence or absence of an anatomic "G-spot." However, few studies have examined the detailed topographic or histologic anatomy of the putative G-spot location.

The "G-Spot" Is Not a Structure Evident on Macroscopic Anatomic Dissection of the Vaginal Wall.

Compared to other neurons of the central nervous system, autonomic preganglionic neurons are unusual because most of their axon lies in the periphery. These axons are vulnerable to injury during surgical procedures, yet in comparison to peripheral neurons and somatic motor neurons, the impact of injury on preganglionic neurons is poorly understood. Here, we have investigated the impact of axotomy on sacral preganglionic neurons, a functionally diverse group of neurons required for micturition, defecation, and sexual function. We have previously observed that after axotomy, the injury-related transcription factor activating transcription factor-3 (ATF3) is upregulated in only half of these neurons (Peddie and Keast, 2011: PMID: 21283532). In the current study, we have investigated if this response is constrained to particular subclasses of preganglionic neurons that have specific functions or signaling properties. Seven days after unilateral pelvic nerve transection, we quantified sacral preganglionic neurons expressing ATF3, many but not all of which co-expressed c-Jun. This response was independent of soma size. Subclasses of sacral preganglionic neurons expressed combinations of somatostatin, calbindin, and neurokinin-1 receptor, each of which showed a similar response to injury. We also found that in contrast to thoracolumbar preganglionic neurons, the heat shock protein-25 (Hsp25) was not detected in naive sacral preganglionic neurons but was upregulated in many of these neurons after axotomy; the majority of these Hsp25 neurons expressed ATF3. Together, these studies reveal the molecular complexity of sacral preganglionic neurons and their responses to injury. The simultaneous upregulation of Hsp25 and ATF3 may indicate a distinct mechanism of regenerative capacity after injury.

Axonal Injury Induces ATF3 in Specific Populations of Sacral Preganglionic Neurons in Male Rats.

Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.

Article	Year
Neurotrophic actions initiated by proNGF in adult sensory neurons may require peri-somatic glia to drive local cleavage to NGF. Journal of neurochemistry\| PubMed ID: 22621370	2012
Co-cultures provide a new tool to probe communication between adult sensory neurons and urothelium. The Journal of urology\| PubMed ID: 23353045	2013
Innervation of developing and adult organs. Organogenesis\| PubMed ID: 23974170	2013
Characterization of bladder sensory neurons in the context of myelination, receptors for pain modulators, and acute responses to bladder inflammation. Frontiers in neuroscience\| PubMed ID: 24223534	2013
Characterization of axons expressing the artemin receptor in the female rat urinary bladder: a comparison with other major neuronal populations. The Journal of comparative neurology\| PubMed ID: 25043933	2014
Regeneration of sensory but not motor axons following visceral nerve injury. Experimental neurology\| PubMed ID: 25725351	2015
Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin. Molecular and cellular neurosciences\| PubMed ID: 25752731	2015
Peripheral injury of pelvic visceral sensory nerves alters GFRα (GDNF family receptor alpha) localization in sensory and autonomic pathways of the sacral spinal cord. Frontiers in neuroanatomy\| PubMed ID: 25914629	2015
An illustrated anatomical ontology of the developing mouse lower urogenital tract. Development (Cambridge, England)\| PubMed ID: 25968320	2015
Developing a functional urinary bladder: a neuronal context. Frontiers in cell and developmental biology\| PubMed ID: 26389118	2015
Renaming all spinal autonomic outflows as sympathetic is a mistake. Autonomic neuroscience : basic & clinical\| PubMed ID: 28566236	2017
The "G-Spot" Is Not a Structure Evident on Macroscopic Anatomic Dissection of the Vaginal Wall. The journal of sexual medicine\| PubMed ID: 29198508	2017
Axonal Injury Induces ATF3 in Specific Populations of Sacral Preganglionic Neurons in Male Rats. Frontiers in neuroscience\| PubMed ID: 30405344	2018
A temporal and spatial map of axons in developing mouse prostate. Histochemistry and cell biology\| PubMed ID: 30976911	2019

Janet R. Keast

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