5-HT3 Signaling Alters Development of Sacral Neural Crest Derivatives That Innervate the Lower Urinary Tract

The autonomic nervous system derives from the neural crest (NC) and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (LUT). During fetal development, sacral NC cells colonize the urogenital sinus to form pelvic ganglia (PG) flanking the bladder neck. The coordinated activity of PG neurons is required for normal urination; however, little is known about the development of PG neuronal diversity. To discover candidate genes involved in PG neurogenesis, the transcriptome profiling of sacral NC and developing PG was performed, and we identified the enrichment of the type 3 serotonin receptor (5-HT3, encoded by Htr3a and Htr3b). We determined that Htr3a is one of the first serotonin receptor genes that is up-regulated in sacral NC progenitors and is maintained in differentiating PG neurons. In vitro cultures showed that the disruption of 5-HT3 signaling alters the differentiation outcomes of sacral NC cells, while the stimulation of 5-HT3 in explanted fetal pelvic ganglia severely diminished neurite arbor outgrowth. Overall, this study provides a valuable resource for the analysis of signaling pathways in PG development, identifies 5-HT3 as a novel regulator of NC lineage diversification and neuronal maturation in the peripheral nervous system, and indicates that the perturbation of 5-HT3 signaling in gestation has the potential to alter bladder function later in life.

Plasticity of neurons in mouse major pelvic ganglia in response to loos of physiological input in cases of nerve injury and spinal cord injury

Autonomic dysfunctions present significant effects on day to day functions of spinal cord-injured patients. Most people with spinal cord injury (SCI) reported a desire to recover autonomic functions such as bladder and sexual functions over regaining of locomotor functions. Lower urinary tract function is one of the autonomic functions that is impaired after SCI. Acute SCI results in areflexic bladder and complete urinary retention while patients with chronic SCI suffer from hyperreflexic bladder, incontinence and inefficient voiding. While extensive SCI research has been done locomotor recovery, relatively limited amount of research has been done on the underlying mechanisms of autonomic dysfunctions associated with SCI. The major pelvic ganglia (MPG) are peripheral ganglia that consist of postganglionic neurons that innervate the urogenital organs and are a part of the neural pathway that controls micturition. In mice, there is one MPG on each side of the animal. MPG receives cholinergic parasympathetic input from the preganglionic neurons in the sacral cord through pelvic nerve and sympathetic cholinergic inputs from those in the lumbosacral cord through the hypogastric nerve. Nicotinic cholinergic transmitter system is the major system involved in ganglionic synaptic transmission at the MPG neurons. The goal of this thesis is to understand the effects of neural injury on nicotinic cholinergic transmission at the postganglionic MPG neurons that innervate the urogenital organs. We are interested in the properties of MPG neurons in these injury states because these neurons are the final neurons in the autonomic pathway that directly innervate the target organs whose functions are compromised as results of these injuries. Before we could study the state of MPG neurons in injury conditions, we first needed to characterize the properties of these neurons in uninjured physiological state. By characterizing the normal cholinergic transmission in control mice, we are able to investigate the changes in the system after injury. Secondly, we sought to understand how MPG neurons respond to abrupt loss of all synaptic inputs. In these studies, we severed all the preganglionic neurons to MPG on one side of the animals leaving the other side intact. Finally, we studied the effects spinal cord injury on the synaptic transmission at the MPG. Spinal cord injury presumably presents altered forms of presynaptic inputs from the spinal cord to the MPG neurons due to hyperreflexic nature of the reflex pathway after SCI. We utilized molecular, electrophysiological, and pharmacological approaches using mouse models to answer our questions. In the first chapter of the thesis, I characterized the synaptic, passive, and firing properties of the MPG neurons in both male and female mice. I also characterized the nicotinic acetylcholine receptor subunits involved in cholinergic neurotransmission at the MPG. In the second chapter, I studied the effects of loss of direct inputs to the MPG neurons in both ipsilateral and contralateral intact ganglia in male mice. I performed unilateral decentralization of inputs to the MPG by severing pelvic and hypogastric nerves. In the third chapter, I studied the effects of spinal cord injury on properties of presynaptic inputs to the MPG as well as postganglionic properties, passive properties and firing properties of MPG neurons. In this injury model, I performed complete transections of the spinal cords between thoracic spinal segments (T10 and T11) in both male and female mice. All the mice had impaired bladder reflexes after spinal transection. Our results showed that decentralization and spinal cord injury effect the synaptic transmission at the MPG as well as the properties of the MPG neurons differently. These effects could be due to influences from both the nature of presynaptic input and the functional state of the target organ. We also observed different effects of spinal cord injury between MPG neurons of males and female. Understanding the mechanisms of changes at the neurotransmission at MPG neurons would be important in developing therapeutic measures for autonomic dysfunctions of the urogenital organs in nerve injury or in spinal cord injury.

Distribution and immunohistochemical characteristics of cocaine- and amphetamineregulated transcript-positive nerve elements in the pelvic ganglia of the female pig

Cocaine- and amphetamine-regulated transcript (CART) peptides are widely expressed not only in the brain but also in numerous endocrine/neuroendocrine cells as well as in neurons of the peripheral nervous system. The present study investigated the distribution patterns of CART-like immunoreactivity in the pelvic plexus (PP) of the female pig. The co-expression of CART with principal neurotransmitter markers: choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), serotonin (5-HT) or biologically active neuropeptides: pituitary adenylate cyclase-activating polypeptide (PACAP), substance P (SP), calbindin was analyzed using double immunohistochemical stainings. Amongst neurons immunopositive to Hu C/D panneuronal marker as many as 4.1 ± 1.2% in right and 4.4 ± 1.6% in left pelvic ganglia were found to express CART. The vast majority of CART-IR ganglionic neurons were predominantly small in size and were evenly scattered throughout particular ganglia. Immunoreactivity to CART was also detected in numerous nerve terminals (which frequently formed pericellular formations around CART-negative perikarya) as well as in numerous nerve fibres within nerve branches interconnecting the unilateral pelvic ganglia. Immunohistochemistry revealed that virtually all CART-IR neurons were cholinergic in nature and CART-IR basket-like formations frequently encircled TH-positive/CART-negative perikarya. None of CART-IR ganglionic neurons showed immunoreactivity to SP, PACAP, 5-HT or calbindin. CART-IR nerve fibres ran in a close vicinity to serotonin-containing cells or faintly labelled SP-expressing neurons. On the other hand, PACAP-IR, SP-IR (but not 5-HT-positive) nerve terminals were found to run in close proximity to CART-IR neurons. Our results indicate that: 1) CART present in PP may influence the activity of pelvic ganglionic neurons/SIF cells, 2) PP should be considered as a potential source of CART-like supply to pelvic viscera and 3) functional interactions between CART and SP or PACAP are possible at the periphery.