S4.3 Neurturin and GDNF effects on maturation and plasticity of urogenital innervation

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Abstracts / Autonomic Neuroscience: Basic and Clinical 149 (2009) 1–126

S4. Neurotrophic factors in autonomic nervous system development

Acknowledgements: This work was supported by the Wellcome Trust.

S4.1 Novel regulators of sympathetic innervation in the developing mouse

doi:10.1016/j.autneu.2009.05.021

A. Davies (School of Biosciences, Biomedical Building, Museum Avenue, Cardiff, CF10 3US, UK), H. Gutierrez (School of Biosciences, Biomedical Building, Museum Avenue, Cardiff, CF10 3US, UK), G. O'Keeffe (School of Biosciences, Biomedical Building, Museum Avenue, Cardiff, CF10 3US, UK), C. Kos (HNRCA at Tufts University, Boston, MA02111, USA), P.P. Pandolfi (Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA02115, USA), D. Riccardi (School of Biosciences, Biomedical Building, Museum Avenue, Cardiff, CF10 3US, UK), C. Riccardi (Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Perugia, Via del Giochetto, 06100 Perugia, Italy), T. Vizard (School of Biosciences, Biomedical Building, Museum Avenue, Cardiff, CF10 3US, UK) Nerve growth factor (NGF) is the paradigmatic target-derived neurotrophic factor on which the foundations of neurotrophic theory are based. Sympathetic neurons depend for survival on a supply of NGF synthesized by their targets and conveyed to their cell soma by retrograde axonal transport. The limiting supply of target-derived NGF not only governs the number of sympathetic neurons that survive during development, it also plays an essential role in promoting the growth and branching of the axon terminals of these neurons within their distal targets. To identify additional extracellular signals that regulate distal target field innervation, we screened mouse superior cervical ganglion (SCG) sympathetic neurons for the expression of a wide variety of receptor classes at a stage in development when the axon terminals of these neurons are branching extensively in their distal targets. Two such receptors whose importance we have demonstrated in regulating sympathetic innervation include a member of the TNF superfamily, GITR, and the G protein-coupled receptor, extracellular calcium-sensing receptor (CaSR). GITR and its ligand GITRL are co-expressed in SCG neurons when their axons are innervating their targets under the influence of target-derived NGF. In culture, GITRL enhances NGF-promoted neurite growth from neonatal SCG neurons, and preventing GITR– GITRL interaction in these neurons or GITR knockdown inhibits NGFpromoted neurite growth without affecting neuronal survival. GITR_/_ neonates have reduced sympathetic innervation density in vivo compared with GITR+/+ littermates. GITR activation is required for the phosphorylation of ERK1/ERK2 by NGF that is necessary for neurite growth. Our results reveal an unsuspected signalling loop in developing sympathetic neurons that is crucial for NGF-dependent axon growth and target innervation [1]. CaSR monitors the systemic extracellular free ionized calcium level ([Ca2+]o) in organs involved in systemic [Ca2+]o homeostasis. We find high levels of the CaSR in perinatal SCG neurons when their axons are innervating and branching extensively in their targets. Manipulating CaSR function in these neurons by varying [Ca2+]o, using CaSR agonists and antagonists or expressing a dominant-negative CaSR markedly affects neurite growth in vitro Sympathetic neurons lacking the CaSR have smaller neurite arbors in vitro, and sympathetic innervation density is reduced in CaSR-deficient mice in vivo. Our findings reveal a crucial role for [Ca2+]o and the CaSR in the establishment of sympathetic innervation during development [2]. [1] O'Keeffe, G., Gutierrez, H., Pandolfi, P. P., Riccardi, C., Davies, A. M. 2008. NGF-promoted axon growth and target innervation requires GITRL-GITR signalling. Nature Neuroscience 11, 135–142. [2] Vizard, T. N., O'Keeffe, G., Gutierrez, H., Kos, C. H., Riccardi, D., Davies, A. M. 2008. Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor. Nature Neuroscience 11, 285–291.

S4.2 Signals and cell behaviors in the development and pathology of the enteric nervous system H. Enomoto (Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan), T. Uesaka (Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan), C. Nishiyama (Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan), H.M. Young (Department of Anatomy and Cell Biology, University of Melbourne, Melbourne, Via, Australia), D.F. Newgreen (The Murdoch Childrens Research Institute, Parkville, Via, Australia) The enteric nervous system (ENS) is the most complex division of the peripheral nervous system, and controls motility and blood flow of the gut which is vital to maintain animal life. The vast majority of cells in the ENS are derived from the enteric neural crest-derived cells (ENCCs) that migrate through the whole length of the gut from the esophagus to the distal end of the colon. Successful colonization of the gut by ENCCs relies on signals from Glial cell line-Derived Neurotrophic Factor (GDNF) via RET/GFRa receptors. In human, mutations affecting RET expression confer susceptibility to Hirschsprung's disease (HSCR), a condition characterized by the absence of the enteric ganglia in the distal colon (intestinal aganglionosis). Elucidating RET function is thus vital for understanding development and pathology of the ENS. To understand the relationship between Ret dosage and organogenesis, we generated an allelic series of Ret and decreased RET expression levels to varying degrees in mice. Mice expressing RET at approximately one third of its normal levels recapitulated clinical and genetic features of HSCR including distal colon aganglionosis and sex-biased incomplete penetrance, demonstrating that this mouse line can serve as a mouse model for HSCR. Analysis of ENS development revealed that a delay in gut colonization by ENCCs and impaired ENCC survival are the two major causes for intestinal aganglionosis in the HSCR model mouse. To assess contribution levels of cell death to the overall aganglionosis phenotype, we engineered the Ret locus to increase expression levels of an anti-cell death gene, Bcl-XL, in ENCCs. Manifestation of intestinal aganglionosis is almost completely suppressed by Ret-Bcl-XL allele in the HSCR model mouse. The results suggest that cell death is principally involved in the etiology of HSCR. Finally, by using mice engineered to express Kikume, a photoconvertible fluorescent protein, in all ENCCs, we demonstrate that ENS in the distal colon is derived from a specific subset of ENCCs. We also show how migratory behaviors of this ENCC population are altered by RET dysfunction, leading to form a basis for HSCR. doi:10.1016/j.autneu.2009.05.022

S4.3 Neurturin and GDNF effects on maturation and plasticity of urogenital innervation J.R. Keast (Pain Management Research Institute and Kolling Institute of Medical Research, University of Sydney at Royal North Shore Hospital, St Leonards, NSW 2065, Australia) Many of the secretory and motor functions of the urogenital tract are controlled by pelvic autonomic ganglia, which contain both sympathetic and parasympathetic neurons. This co-location of

Abstracts / Autonomic Neuroscience: Basic and Clinical 149 (2009) 1–126

chemically and functionally distinct pathways that receive inputs from different spinal levels leads to interesting questions regarding the cues that control their development, maintenance and responses to injury. In addition, these reflex circuits change dramatically after birth, when voluntary control of voiding develops and later when rising levels of gonadal hormones drive the maturation of neuronal circuits involved in reproductive behaviours. We have begun to investigate the role of the GDNF (glial cell linederived neurotrophic factor) family ligands in the early postnatal period, to understand the mechanisms driving establishment of these pelvic autonomic nerve circuits. We have also performed studies in adults to determine if some of these mechanisms are recapitulated after injury. This presentation will discuss how neurturin and GDNF regulate differentiation and projections of pelvic ganglion neurons, specifically in relation to parasympathetic control of urogenital functions. It will also propose a role of these neurotrophic factors during adulthood, not only in the context of stimulating regenerative growth but also participating in aberrant growth or compensatory responses within these circuits. The presentation will include our recent findings that implicate GDNF and artemin as key mediators of structural plasticity within bladder sensory and motor nerves. These studies may provide new strategies for restoring function after injury and modulating aberrant behaviour in disease states within the urogenital tract.

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These data were confirmed immunocytochemically in vivo, as intestinal inflammation was accompanied not only by increased numbers of spinal neuronal somata expressing substance P and CGRP, but also by enhanced expression levels of SOM, SSTR1, SSTR2A and SSTR4 in spinal ganglia. These data reveal that intestinal inflammation induces the onset of both pro-inflammatory cascades and endogenous systems destined to prevent excessive tissue damage. Moreover, functional and morphological evidence is provided for the first time that SOM is able to directly modulate intestinal inflammatory responses by interference with the mast cell–neuron communication. Finally, the observation that SOM affects HIS- and 5-HT-induced responses in non-neuronal cells as well, suggests that SOM could indirectly modulate sensory neuronal characteristics through modulation of the surrounding non-neuronal cells. doi:10.1016/j.autneu.2009.05.024

S5.2 Reactions of enteric and sympathetic ganglia to a brief inflammatory stimulus in the guinea-pig ileum

J. Van Op den bosch (Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium), L. Van Nassauw (Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium; Laboratory of Human Anatomy and Embryology, University of Antwerp, Antwerp, Belgium), E. Van Marck (Laboratory of Pathology, University of Antwerp, Antwerp, Belgium), J.-P. Timmermans (Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium)

J.B. Furness (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia), M. Thacker (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia), B. Hunne (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia), L. Pontell (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia), H.C. Abeysinghe (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia), P. Castelucci (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Department of Anatomy, University of São Paulo, São Paulo, Brazil), M. Bagyánszki (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Department of Physiology, Anatomy and Neuroscience, University of Szeged, Hungary), K. Nurgali (Department of Physiology, University of Melbourne, Parkville, Victoria 3010, Australia), R. Bron (Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3010, Australia)

Intestinal inflammatory responses are coordinated by the extensive bidirectional communication between mast cells and enteric neurons embedded within the intestinal wall. However, despite the requirement of endogenous mediators able to inhibit this selfreinforcing communication network, no such inhibitory mediators have been identified so far. Recently, the expression of multiple somatostatin receptor (SSTR) subtypes was demonstrated in mucosal mast cells and in extrinsic and intrinsic nerve fibres in the intestinal wall. These findings suggest that somatostatin (SOM) is able to modulate the inflammatory activities of both neurons and mast cells. Therefore, we assessed the modulatory effects of SOM on the shortterm and long-term effects induced by two main mast cell mediators, histamine (HIS) and 5-HT, on sensory neuronal characteristics using murine spinal neuronal cultures. Using ELISA and calcium live cell imaging, HIS and 5-HT were shown to acutely induce neuronal CGRP release and calciummediated activation of both neurons and non-neuronal cells. Both of these mast cell-induced effects were strongly reduced by preincubating the spinal cultures with SOM. Long-term incubation of the spinal cultures with HIS and 5-HT significantly elevated neuronal expression of substance P, CGRP and SOM as demonstrated by qPCR and quantitative analysis. Also these long-term effects of HIS and 5-HT were partly inhibited by chronic SOM exposure. Immunocytochemical and molecular–biological experiments suggest the involvement of SSTR1 and SSTR2A in these SOM-dependent effects.

Clinical investigations indicate that a brief period of severe gastroenteritis can cause a long-lasting complex of functional abnormalities of the gastrointestinal tract, post-inflammatory irritable bowel syndrome (IBS). Post-inflammatory IBS has been modelled in animals in recent years, and in particular hyperexcitability of neurons in enteric and dorsal root ganglia have been investigated. More recently, we have observed increased excitability of sympathetic neurons that innervate the intestine, following inflammation of the distal ileum [1]. In the current study, we have investigated the histological changes and proliferation of eosinophils and T-cells at 3 h to 56 days after ileitis that was induced by injection of TNBS (2,4,6trinitrobenzenesulfonic acid) into the lumen of the distal ileum. For the 7 day time point, we have also investigated changes in the electrophysiological properties of enteric and celiac ganglion neurons. At 3 h, the mucosal surface was completely ablated and villi were destroyed, although mucosal glands remained intact. By 6 h an epithelial covering was substantially restored, and by 1 day there was full re-epithelialisation. During the restoration, the epithelium developed from a squamous cell covering, without villi or goblet cells, to a fully differentiated columnar epithelium containing a normal complement of goblet cells and with mature villi at about 7 days. Prominent phagocytic activity of enterocytes, which is not normally observed, occurred at 1 day to 7 days after TNBS. Intraepithelial immune cells were also found. We observed an apparent expulsion of lysosome-like particles into the lumen, indicating that

doi:10.1016/j.autneu.2009.05.023

S5: Inflammation and sensory mechanisms S5.1 Somatostatin modulates mast cell-induced responses in murine spinal neurons and satellite cells