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Visualizing the enteric nervous system using genetically engineered double reporter mice: Comparison with immunofluorescence

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      Abstract

      Background and aimsThe enteric nervous system (ENS) plays a crucial role in the control of gastrointestinal motility, secretion and absorption functions. Immunohistochemistry has been widely used to visualize neurons of the ENS for more than two decades. Genetically engineered mice that report specific proteins can also be used to visualize neurons of the ENS. The goal of our study was to develop a mouse that expresses fluorescent neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT), the two proteins expressed in 95% of the ENS neurons. We compared ENS neurons visualized in the reporter mouse with the wild type mouse stained using classical immunostaining techniques.MethodsMice hemizygous for ChAT-ChR2-YFP BAC transgene with expression of the mhChR2:YFP fusion protein directed by ChAT promoter/enhancer regions on the BAC transgene were purchased commercially. The Cre/LoxP technique of somatic recombination was used to construct mice with nNOS positive neurons. The two mice were crossbred and tissues were harvested and examined using fluorescent microscopy. Immunostaining was performed in the wild type mice, using antibodies to nNOS, ChAT, Hu and PGP 9.5.ResultsGreater than 95% of the ENS neurons were positive for either nNOS or ChAT or both. The nNOS and ChAT neurons and their processes in the ENS were well visualized in all the regions of the GI tract, i.e., esophagus, small intestine and colon. The number of nNOS and ChAT neurons was approximately same in the reporter mouse and immunostaining method in the wild type mouse. The nNOS fluorescence in the reporter mouse was seen in both cytoplasm as well as nucleus but in the immunostained specimens it was seen only in the cytoplasm.ConclusionWe propose that the genetically engineered double reporter mouse for ChAT and nNOS proteins is a powerful tool to study of the effects of various diseases on the ENS without the need for immunostaining.

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      Most cited references 17

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      A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.

      The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.
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        The enteric nervous system and gastrointestinal innervation: integrated local and central control.

        The digestive system is innervated through its connections with the central nervous system (CNS) and by the enteric nervous system (ENS) within the wall of the gastrointestinal tract. The ENS works in concert with CNS reflex and command centers and with neural pathways that pass through sympathetic ganglia to control digestive function. There is bidirectional information flow between the ENS and CNS and between the ENS and sympathetic prevertebral ganglia.The ENS in human contains 200-600 million neurons, distributed in many thousands of small ganglia, the great majority of which are found in two plexuses, the myenteric and submucosal plexuses. The myenteric plexus forms a continuous network that extends from the upper esophagus to the internal anal sphincter. Submucosal ganglia and connecting fiber bundles form plexuses in the small and large intestines, but not in the stomach and esophagus. The connections between the ENS and CNS are carried by the vagus and pelvic nerves and sympathetic pathways. Neurons also project from the ENS to prevertebral ganglia, the gallbladder, pancreas and trachea.The relative roles of the ENS and CNS differ considerably along the digestive tract. Movements of the striated muscle esophagus are determined by neural pattern generators in the CNS. Likewise the CNS has a major role in monitoring the state of the stomach and, in turn, controlling its contractile activity and acid secretion, through vago-vagal reflexes. In contrast, the ENS in the small intestine and colon contains full reflex circuits, including sensory neurons, interneurons and several classes of motor neuron, through which muscle activity, transmucosal fluid fluxes, local blood flow and other functions are controlled. The CNS has control of defecation, via the defecation centers in the lumbosacral spinal cord. The importance of the ENS is emphasized by the life-threatening effects of some ENS neuropathies. By contrast, removal of vagal or sympathetic connections with the gastrointestinal tract has minor effects on GI function. Voluntary control of defecation is exerted through pelvic connections, but cutting these connections is not life-threatening and other functions are little affected.
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          Anatomy and physiology of the enteric nervous system.

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            Author and article information

            Affiliations
            [1 ]Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California
            [2 ]San Diego VA Health Care Systemm, San Diego, California
            Wayne State University, UNITED STATES
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            • Conceptualization: RKM.

            • Data curation: YJ HD.

            • Formal analysis: YJ RKM.

            • Funding acquisition: RKM.

            • Investigation: RKM YJ LE EH KCI.

            • Methodology: LE YJ RKM HD.

            • Project administration: RKM.

            • Supervision: RKM.

            • Writing – original draft: RKM YJ.

            • Writing – review & editing: RKM YJ LE.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, CA USA )
            1932-6203
            3 February 2017
            2017
            : 12
            : 2
            28158225
            5291392
            10.1371/journal.pone.0171239
            PONE-D-16-24759
            (Editor)

            This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

            Counts
            Figures: 8, Tables: 1, Pages: 12
            Product
            Funding
            Funded by: funder-id http://dx.doi.org/10.13039/100009012, Veterans Affairs San Diego Healthcare System;
            Award ID: MERITS
            Award Recipient :
            Funded by VA MERIT Grant to RKM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology and Life Sciences
            Cell Biology
            Cellular Types
            Animal Cells
            Neurons
            Biology and Life Sciences
            Neuroscience
            Cellular Neuroscience
            Neurons
            Research and Analysis Methods
            Specimen Preparation and Treatment
            Staining
            Immunostaining
            Biology and Life Sciences
            Cell Biology
            Cellular Structures and Organelles
            Cytoplasm
            Biology and Life Sciences
            Neuroscience
            Sensory Perception
            Vision
            Biology and Life Sciences
            Psychology
            Sensory Perception
            Vision
            Social Sciences
            Psychology
            Sensory Perception
            Vision
            Biology and Life Sciences
            Anatomy
            Digestive System
            Gastrointestinal Tract
            Esophagus
            Medicine and Health Sciences
            Anatomy
            Digestive System
            Gastrointestinal Tract
            Esophagus
            Research and analysis methods
            Specimen preparation and treatment
            Staining
            Nuclear staining
            DAPI staining
            Research and Analysis Methods
            Specimen Preparation and Treatment
            Staining
            Cytoplasmic Staining
            Biology and Life Sciences
            Biochemistry
            Neurochemistry
            Neurochemicals
            Nitric Oxide
            Biology and Life Sciences
            Neuroscience
            Neurochemistry
            Neurochemicals
            Nitric Oxide
            Custom metadata
            All relevant data are within the paper.

            Uncategorized

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