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      Age-related gene expression analysis in enteric ganglia of human colon after laser microdissection

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          Abstract

          The enteric nervous system (ENS) poses the intrinsic innervation of the gastrointestinal tract and plays a critical role for all stages of postnatal life. There is increasing scientific and clinical interest in acquired or age-related gastrointestinal dysfunctions that can be manifested in diseases such as gut constipation or fecal incontinence. In this study, we sought to analyze age-dependent changes in the gene expression profile of the human ENS, particularly in the myenteric plexus. Therefore, we used the laser microdissection technique which has been proven as a feasible tool to analyze distinct cell populations within heterogeneously composed tissues. Full biopsy gut samples were prepared from children (4–12 months), middle aged (48–58 years) and aged donors (70–95 years). Cryosections were histologically stained with H&E, the ganglia of the myenteric plexus identified and RNA isolated using laser microdissection technique. Quantitative PCR was performed for selected neural genes, neurotransmitters and receptors. Data were confirmed on protein level using NADPH-diaphorase staining and immunohistochemistry. As result, we demonstrate age-associated alterations in site-specific gene expression pattern of the ENS. Thus, in the adult and aged distal parts of the colon a marked decrease in relative gene expression of neural key genes like NGFR, RET, NOS1 and a concurrent increase of CHAT were observed. Further, we detected notable regional differences of RET, CHAT, TH, and S100B comparing gene expression in aged proximal and distal colon. Interestingly, markers indicating cellular senescence or oxidative stress ( SNCA, CASP3, CAT, SOD2, and TERT) were largely unchanged within the ENS. For the first time, our study also describes the age-dependent expression pattern of all major sodium channels within the ENS. Our results are in line with previous studies showing spatio-temporal differences within the mammalian ENS.

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          Most cited references61

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          Types of neurons in the enteric nervous system.

          This paper, written for the symposium in honour of more than 40 years' contribution to autonomic research by Professor Geoffrey Burnstock, highlights the progress made in understanding the organisation of the enteric nervous system over this time. Forty years ago, the prevailing view was that the neurons within the gut wall were post-ganglionic neurons of parasympathetic pathways. This view was replaced as evidence accrued that the neurons are part of the enteric nervous system and are involved in reflex and integrative activities that can occur even in the absence of neuronal influence from extrinsic sources. Work in Burnstock's laboratory led to the discovery of intrinsic inhibitory neurons with then novel pharmacology of transmission, and precipitated investigation of neuron types in the enteric nervous system. All the types of neurons in the enteric nervous system of the small intestine of the guinea-pig have now been identified in terms of their morphologies, projections, primary neurotransmitters and physiological identification. In this region there are 14 functionally defined neuron types, each with a characteristic combination of morphological, neurochemical and biophysical properties. The nerve circuits underlying effects on motility, blood flow and secretion that are mediated through the enteric nervous system are constructed from these neurons. The circuits for simple motility reflexes are now known, and progress has been made in analysing those involved in local control of blood flow and transmucosal fluid movement in the small intestine.
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            Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice.

            Sodium currents and action potentials were characterized in Purkinje neurons from ataxic mice lacking expression of the sodium channel Scn8a. Peak transient sodium current was approximately 60% of that in normal mice, but subthreshold sodium current was affected much more. Steady-state current elicited by voltage ramps was reduced to approximately 30%, and resurgent sodium current, an unusual transient current elicited on repolarization following strong depolarizations, was reduced to 8%-18%. In jolting mice, with a missense mutation in Scn8a, steady-state and resurgent current were also reduced, with altered voltage dependence and kinetics. Both spontaneous firing and evoked bursts of spikes were diminished in cells from null and jolting mice. Evidently Scn8a channels carry most subthreshold sodium current and are crucial for repetitive firing.
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              Existence of distinct sodium channel messenger RNAs in rat brain.

              The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel) and from chick cardiac muscle consist of a single polypeptide of relative molecular mass (Mr) approximately 260,000 (260K), whereas those purified from rat brain and skeletal muscle contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37-45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.
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                Author and article information

                Contributors
                Journal
                Front Aging Neurosci
                Front Aging Neurosci
                Front. Aging Neurosci.
                Frontiers in Aging Neuroscience
                Frontiers Media S.A.
                1663-4365
                15 October 2014
                2014
                : 6
                : 276
                Affiliations
                [1] 1CELLT Research Group, Translational Centre for Regenerative Medicine, University of Leipzig Leipzig, Germany
                [2] 2Department of General and Visceral Surgery, St. George's Hospital Leipzig, Germany
                [3] 3Department of Tissue Engineering and Regenerative Medicine, University Hospital Wuerzburg Wuerzburg, Germany
                [4] 4Division of Molecular biological-biochemical Processing Technology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig Leipzig, Germany
                [5] 5Department of Pediatric Surgery, University of Leipzig Leipzig, Germany
                Author notes

                Edited by: Rodrigo Orlando Kuljiš, Zdrav Mozak Limitada, Chile

                Reviewed by: Eric Blalock, University of Kentucky, USA; Helmut Heinsen, University of Wuerzburg, Germany

                *Correspondence: Marco Metzger, Department of Tissue Engineering and Regenerative Medicine, Fraunhofer Project Group: Regenerative Technologies for Oncology, University Hospital Wuerzburg, Roentgenring 11, 97070 Wuerzburg, Germany e-mail: marco.metzger@ 123456igb.fraunhofer.de

                This article was submitted to the journal Frontiers in Aging Neuroscience.

                †Present address: Susan Hetz, Clinic-oriented Therapy Assessment Unit, Department of Cell Therapy, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany;

                Roman Metzger, Department of Pediatrics and Adolescent Medicine, Salzburg County Hospital, Salzburg, Austria;

                Marco Metzger, Department of Tissue Engineering and Regenerative Medicine, Fraunhofer Project Group: Regenerative Technologies for Oncology, University Hospital Wuerzburg, Wuerzburg, Germany

                ‡These authors have contributed equally to this work.

                Article
                10.3389/fnagi.2014.00276
                4197768
                25360110
                631f4ada-c857-4c51-8cb8-079a6861d253
                Copyright © 2014 Hetz, Acikgoez, Moll, Jahnke, Robitzki, Metzger and Metzger.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 03 February 2014
                : 24 September 2014
                Page count
                Figures: 4, Tables: 4, Equations: 0, References: 62, Pages: 12, Words: 8061
                Categories
                Neuroscience
                Original Research Article

                Neurosciences
                laser microdissection,enteric nervous system,myenteric plexus,aging,sodium channels
                Neurosciences
                laser microdissection, enteric nervous system, myenteric plexus, aging, sodium channels

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