1 Assistant Professor Neuroscience Program and Department of Physiology Michigan State University East Lansing, Michigan USA The requirement to nourish and sustain an organism, as well as to defend it from pathogens and toxins, food antigens, environmental irritants, parasites and infectious agents places a special burden on the GI tract The gut has to move food along its length, digest this food and absorb nutrients as well as protect itself from this process It takes a brain to organize, initiate and detect these processes 2 The gut and its brain The enteric nervous system (ENS): A continuous ganglionated network extending the length of the gut Contains a similar number of neurons as the spinal cord (or the brain of a cat) Esophagus Stomach Small intestine Large intestine Really?? Submucosal ganglia 3 Myenteric ganglia 1
Normal gut function requires the enteric nervous system (ENS) +TTX 4 Video courtesy of Dr. Gary Mawe, University of Vermont Control of gut motility 1) Myogenic 3) Nervous 2) Hormonal 5 M.B. Hansen, Physiol. Res. (2003) The organization of the ENS of human and medium large mammals Myenteric Plexus Submucosal Plexus Furness, J. B. (2012)The enteric nervous system and neurogastroenterology Nat. Rev. Gastroenterol. Hepatol.doi:10.1038/nrgastro.2012.32 Modified with permission from Furness, J.B. The Nervous System (Blackwell, Oxford, 2006) 6 2
The enteric nervous system Submucosal plexus: between the mucosa and circular muscle controls absorption/secretion Myenteric plexus: between circular and longitudinal muscle controls motility Lumen 7 Intrinsic reflex circuits that control motility involve enteroendocrine and neuronal signaling Oral Longitudinal muscle Myenteric plexus Anal Circular muscle Submucosal plexus Contraction Relaxation Mucosa 8 Pressure gradient surround neurons in the enteric nervous system Extrinsic (central) neural input Intrinsic (enteric) neural input neuron 9 3
10 Types of enteric glial cells a: type I b: type II d: type III d: type IV Gulbransen, B. D. & Sharkey, K. A. (2012)Novel functional roles for enteric glia in the gastrointestinal tract Nat. Rev. Gastroenterol. Hepatol.doi:10.1038/nrgastro.2012.138 11 What do glia do? 1. Insulate and provide metabolic support to neurons 2. Regulate vasculature 3. Housekeeping functions (maintain the extracellular milieu) 4. Active roles 12 Modified from original artwork of Santiago Ramón y Cajal 4
supply neurons with essential precursors GS Glutamine Glutamate Glutamate/GABA NO 13 What do glia do? 1. Insulate and provide metabolic support to neurons 2. Regulate vasculature 3. Housekeeping functions (maintain the extracellular milieu) 4. Active roles 14 Modified from original artwork of Santiago Ramón y Cajal 3-D imaging, illustration, and quantitation of enteric glial network in transparent human colon mucosa Liu et al. Neurogastroenterology & Motility Volume 25, Issue 5, pages e324-e338, 17 MAR 2013 DOI: 10.1111/nmo.12115 http://onlinelibrary.wiley.com/doi/10.1111/nmo.12115/full#nmo12115-fig-0006 15 5
Contacts between myenteric glia and periganglionic capillaries GFAP + capillaries Fu Y et al. Am J Physiol Gastrointest Liver Physiol 2013;304:G1-G11 16 2013 by American Physiological Society What do glia do? 1. Insulate and provide metabolic support to neurons 2. Regulate vasculature 3. Housekeeping functions (maintain the extracellular milieu) 4. Active roles 17 Modified from original artwork of Santiago Ramón y Cajal regulate availability of neurotransmitters/neuromodulators GS Glutamine Glutamate NO 18 Glutamate/GABA 6
What do glia do? 1. Insulate and provide metabolic support to neurons 2. Regulate vasculature 3. Housekeeping functions (maintain the extracellular milieu) 4. Active roles 19 Modified from original artwork of Santiago Ramón y Cajal Astrocytes modulate synaptic transmission Ca 2+ 20 Purines are important signaling molecules for glia Astrocyte Astrocyte NTPDases Adenosine P2Rs Neuron 21 7
Purines are important signaling molecules in the ENS Longitudinal muscle Myenteric plexus Excitatory motor neuron Inhibitory motor neuron Circular muscle SP ACh NO Submucosalplexus Mucosa 22 Oral Anal Vesiculated nerve processes in contact with enteric glia show pre-synaptic specializations Axon Axon Glia Glia 23 Modified from Gabella 1972 J. Anat Innervation of EGCs and their processes A single EGC (G) and its nearby varicosities (V) using an inverted image, and highlighting the varicosities on the same axon with color as well as the EGC 24 Broadhead M J et al. J Physiol 2012;590:335-350 8
have the potential to respond to neurotransmitters/neuromodulators Norepinephrine P2Y1, P2Y2, P2Y4, P2X7 Acetylcholine mglur5 Glutamate 25 Calcium (Ca 2+ ) imaging Ca 2+ Ca 2+ Ca 2+ Ca IP 2+ 3 Store 26 Do enteric glia respond to? Ca 2+ PLCβ2 Kimball & Mulholland J. Neurochem. 1996 27 9
Intercellular calcium waves in cultured enteric glia from neonatal guinea pig Ca 2+ Ca 2+ +U73122 Ca 2+ Glia Volume 42, Issue 3, pages 252-262, 25 MAR 2003 DOI: 10.1002/glia.10215 http://onlinelibrary.wiley.com/doi/10.1002/glia.10215/full#fig5 28 Intercellular calcium waves in cultured enteric glia from neonatal guinea pig (2) Ca 2+ Ca 2+ +Apyrase Ca 2+ Glia Volume 42, Issue 3, pages 252-262, 25 MAR 2003 DOI: 10.1002/glia.10215 http://onlinelibrary.wiley.com/doi/10.1002/glia.10215/full#fig5 29 stimulates enteric glia in situ 30 Gulbransen and Sharkey Gastroenterology 2009 Apr 136(4): 1349-58 10
-stimulates glial Ca 2+ responses [Ca 2+ ] i High 31 Gulbransen and Sharkey Gastroenterology 2009 Apr 136(4): 1349-58 Low stimulates guinea pig enteric glia in situ 32 Gulbransen and Sharkey Gastroenterology 2009 Apr 136(4): 1349-58 Do enteric glia sense released from enteric neurons?? 33 11
-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice Neurogastroenterology & Motility Volume 21, Issue 8, pages 870-e62, 30 MAR 2009 DOI: 10.1111/j.1365-2982.2009.01302.x http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2982.2009.01302.x/full#f5 34 Do enteric glia respond to neuronal activity? Glia Neurotransmitter FTS Fiber Tract Stimulation 35 FTS-stimulates glial Ca 2+ responses [Ca 2+ ] i High 36 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 Low 12
EFS-stimulates glial Ca 2+ responses (2) FTS 37 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 respond to released from neurons through P2Y receptors PPADS Ca 2+ P2R PLCβ2 TTX U73122 38 Pharmacological agonist profiling Gulbransenand Sharkey Gastroenterology (2009) Postsynaptic neuron Ca 2+ transients in EGCs following anal mucosal stimulation A B 39 Broadhead M J et al. J Physiol 2012;590:335-350 2012 by The Physiological Society 13
Which neuronal component(s) of the ENS talk to glia? Primary afferent Glia Sympathetic Parasympathetic neuron 40 FTS fiber tract stimulation Which neuronal component(s) of the ENS signals to glia? Primary afferent Sympathetic Parasympathetic neuron 41 Nicotinic stimulation of enteric neurons Ave glial resp Ave nn resp 42 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 14
Which neuronal component(s) of the ENS signals to glia? (2) Primary afferent Sympathetic Parasympathetic Hexamethonium neuron 43 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 Which neuronal component(s) of the ENS signals to glia? (3) Capsaicin Primary afferent Sympathetic Parasympathetic neuron 44 TRPV1 stimulation of primary afferents Ave glial resp Ave nn resp 45 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 15
Which neuronal component(s) of the ENS signals to glia? (4) 6-OHDA Primary afferent Sympathetic Parasympathetic neuron 46 **Main extrinsic innervation to GP distal colon is sympathetic 6-OHDA-mediated destruction of tyrosine hydroxylase (TH) immunoreactive nerves Control 6-OHDA 100 um 100 um 47 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 Which neuronal component(s) of the ENS signals to glia? (5) Primary afferent Sympathetic Parasympathetic neuron 48 **Main extrinsic innervation to GP distal colon is sympathetic 16
Total extrinsic denervation Control Denervated Primary afferent Sympathetic 49 Parasympathetic Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 neuron Which neuronal component(s) of the ENS signals to glia? (6) Primary afferent Sympathetic Parasympathetic neuron 50 **Main extrinsic innervation to GP distal colon is sympathetic FTS-mediated glial activation is frequency dependent 51 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 17
The sympathetic co-transmitters norepinephrine, somatostatin, and NPY do not stimulate Ca 2+ responses in enteric glia 52 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 Model of neuron-glia signaling in the colonic myenteric plexus of the guinea pig 53 Gulbransen BD, Bains JS, and Sharkey KA The Journal of Neuroscience; May 12, 2010 neuron to glia communication Neuron Neuron Glia Glia Glia High 54 [Ca 2+ ]i Low 18
Activating neuronal P2X7Rs releases, activating surrounding enteric glia 55 Gulbransenet al. Nature Medicine (2012) Scale = 10% F/F and 30 s released from enteric neurons is converted to ADP and stimulates glial P2Y1Rs Bz P2Y1 enteric neuron glia 56 Gulbransen et al. Nature Medicine (2012) P2X7-stimulated release from enteric neurons depends on pannexin-1 Bz P2Y1 57 neuron Gulbransenet al. Nature Medicine (2012) 19
Glial ectonucleotidases E. Lavoie and B. Gulbransenet al. AJP GastrointestLiver Physiol(2011) 58 neuron ADP monitor purines released by neurons in the myenteric plexus Ca 2+ ADP neuron 59 Gulbransenet al. Nature Medicine (2012) Gulbransenand Sharkey Gastroenterology (2009) Gulbransenet al. The Journal of Neuroscience (2010) Active funding: Acknowledgements NIH/PHS K12HD065879-03 ANMS Research Grant MSU neuroscience startup Keith Sharkey, Ph.D. and the Sharkey Lab University of Calgary Collaborators: The Gulbransen lab: Vladimir Parpura Research technician: University of Alabama, Birmingham Jonathon McClain Jean Sévigny Undergraduate researchers: University of Laval, QC, Canada Rebecca Bubenheimer Robert Vanderkamp Visit us on the web at: Graduate students: https://www.msu.edu/~gulbrans/ Isola Brown 60 20
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