Electrophysiological and morphological features of myenteric neurons of human colon revealed by intracellular recording and dye fills.
electrophysiology
enteric nervous system
immunohistochemistry
morphology
myenteric plexus
Journal
Neurogastroenterology and motility
ISSN: 1365-2982
Titre abrégé: Neurogastroenterol Motil
Pays: England
ID NLM: 9432572
Informations de publication
Date de publication:
04 2023
04 2023
Historique:
revised:
27
12
2022
received:
10
11
2022
accepted:
11
01
2023
pubmed:
7
2
2023
medline:
22
3
2023
entrez:
6
2
2023
Statut:
ppublish
Résumé
Ex vivo intracellular recordings and dye fills, combined with immunohistochemistry, are a powerful way to analyze the enteric nervous system of laboratory animals. Myenteric neurons were recorded in isolated specimens of human colon. A key determinant of successful recording was near-complete removal of circular muscle from the surface of ganglia. Treatment with a collagenase/neutral protease mix before dissection significantly improved recording success and reduced damage to the plexus. Carboxyfluorescein in microelectrodes allowed recorded neurons to be routinely labeled, analyzed, and subjected to multi-layer immunohistochemistry. Carboxyfluorescein revealed morphological details that were not detected by immunohistochemical methods. Of 54 dye-filled myenteric neurons (n = 22), 45 were uni-axonal and eight were multi-axonal. There was a significant bias toward recordings from large neural somata. The close association between morphology and electrophysiology (long after-hyperpolarizations and fast EPSPs) seen in mice and guinea pigs did not hold for human myenteric neuron recordings. No slow EPSPs were recorded; however, disruption to the myenteric plexus during dissection may have led the proportion of cells receiving synaptic potentials to be underestimated. Neurons immunoreactive for nitric oxide synthase were more excitable than non-immunoreactive neurons. Distinctive grooves were observed on the serosal and/or mucosal faces of myenteric neurons in 3D reconstructions. These had varicose axons running through them and may represent a preferential site of synaptic inputs. Human enteric neurons share many features with laboratory animals, but the combinations of features in individual cells appear more variable.
Sections du résumé
BACKGROUND
Ex vivo intracellular recordings and dye fills, combined with immunohistochemistry, are a powerful way to analyze the enteric nervous system of laboratory animals.
METHODS
Myenteric neurons were recorded in isolated specimens of human colon. A key determinant of successful recording was near-complete removal of circular muscle from the surface of ganglia.
KEY RESULTS
Treatment with a collagenase/neutral protease mix before dissection significantly improved recording success and reduced damage to the plexus. Carboxyfluorescein in microelectrodes allowed recorded neurons to be routinely labeled, analyzed, and subjected to multi-layer immunohistochemistry. Carboxyfluorescein revealed morphological details that were not detected by immunohistochemical methods. Of 54 dye-filled myenteric neurons (n = 22), 45 were uni-axonal and eight were multi-axonal. There was a significant bias toward recordings from large neural somata. The close association between morphology and electrophysiology (long after-hyperpolarizations and fast EPSPs) seen in mice and guinea pigs did not hold for human myenteric neuron recordings. No slow EPSPs were recorded; however, disruption to the myenteric plexus during dissection may have led the proportion of cells receiving synaptic potentials to be underestimated. Neurons immunoreactive for nitric oxide synthase were more excitable than non-immunoreactive neurons. Distinctive grooves were observed on the serosal and/or mucosal faces of myenteric neurons in 3D reconstructions. These had varicose axons running through them and may represent a preferential site of synaptic inputs.
CONCLUSIONS
Human enteric neurons share many features with laboratory animals, but the combinations of features in individual cells appear more variable.
Substances chimiques
6-carboxyfluorescein
3301-79-9
Fluoresceins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
e14538Subventions
Organisme : NIH HHS
ID : OT2 OD024899
Pays : United States
Informations de copyright
© 2023 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.
Références
Brehmer A. Structure of enteric neurons. 186 advances in anatomy embryology and cell biology. Springer-Verlag; 2006.
Brehmer A. Classification of human enteric neurons. Histochem Cell Biol. 2021;156(2):95-108. doi:10.1007/s00418-021-02002-y
Dogiel AS. Über den bau der ganglien in den geflechten des darmes und der gallenblase des menschen und der säugethiere. Arch Anat Physiol Leipzig, Anat Abt. Biology. 1899;Jg 1899:130-158.
Costa M, Brookes SJH, Steele PA, Gibbins I, Burcher E, Kandiah CJ. Neurochemical classification of myenteric neurons in the Guinea-pig ileum. Neuroscience. 1996;75(3):949-967.
Hirst GDS, Holman ME, Spence I. Two types of neurones in the myenteric plexus of duodenum in the Guinea-pig. J Physiol (Lond). 1974;236:303-326.
Nishi S, North RA. Intracellular recording from the myenteric plexus of the Guinea-pig ileum. J Physiol (Lond). 1973;231(3):471-491.
Brookes SJH. Retrograde tracing of enteric neuronal pathways. Neurogastroenterol Motil. 2001;13(1):1-18.
Drokhlyansky E, Smillie CS, Van Wittenberghe N, et al. The human and mouse enteric nervous system at single-cell resolution. Research support, N.I.H., extramural research support, non-U.S. Gov't. Cell. 2020;182(6):1606-1622.e23.
May-Zhang AA, Tycksen E, Southard-Smith AN, et al. Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes In situ. Gastroenterology. 2021;160(3):755-770.e26.
Morarach K, Mikhailova A, Knoflach V, et al. Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing. Research support, non-U.S. Gov't. Nat Neurosci. 2021;24(1):34-46.
Zeisel A, Hochgerner H, Lonnerberg P, et al. Molecular architecture of the mouse nervous system. Research support, non-U.S. Gov't. Cell. 2018;174(4):999-1014.e22.
Wattchow DA, Smolilo D, Hibberd T, et al. The human enteric nervous system. Historical and modern advances. Collaboration between science and surgery. Review. ANZ J Surg. 2022;11:11.
Osorio N, Delmas P, Jones PA. Patch clamp recording from enteric neurons in situ. Research support, non-U.S. Gov't. Nat Protoc. 2011;6(1):15-27.
Bornstein JC, Costa M, Furness JB, Lees GM. Electrophysiology and enkephalin immunoreactivity of identified myenteric plexus neurones of Guinea-pig small intestine. J Physiol (Lond). 1984;351:313-325.
Brookes SJ, Meedeniya AC, Jobling P, Costa M. Orally projecting interneurones in the Guinea-pig small intestine. J Physiol (Lond). 1997;505(Pt 2):473-491.
Hibberd TJ, Spencer NJ, Zagorodnyuk VP, Chen BN, Brookes SJ. Targeted electrophysiological analysis of viscerofugal neurons in the myenteric plexus of Guinea-pig colon. Neuroscience. 2014;275:272-284. doi:10.1016/j.neuroscience.2014.04.066
Brookes SJH, Ewart WR, Wingate DL. Intracellular recordings from myenteric neurones in the human colon. J Physiol (Lond). 1987;390(305):305-318.
Carbone SE, Jovanovska V, Brookes SJ, Nurgali K. Electrophysiological and morphological changes in colonic myenteric neurons from chemotherapy-treated patients: a pilot study. Research support, non-U.S. Gov't. Neurogastroenterology & Motility. 2016;28(7):975-984.
Carbone SE, Jovanovska V, Nurgali K, Brookes SJ. Human enteric neurons: morphological, electrophysiological, and neurochemical identification. Research support, non-U.S. Gov't. Neurogastroenterol Motil. 2014;26(12):1812-1816.
Gendusa R, Scalia CR, Buscone S, Cattoretti G. Elution of high-affinity (>10-9 KD) antibodies from tissue sections: clues to the molecular mechanism and use in sequential immunostaining. J Histochem Cytochem. 2014;62(7):519-531. doi:10.1369/0022155414536732
Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-675. doi:10.1038/nmeth.2089
Lomax AE, Sharkey KA, Bertrand PP, Low AM, Bornstein JC, Furness JB. Correlation of morphology, electrophysiology and chemistry of neurons in the myenteric plexus of the Guinea-pig distal colon. J Auton Nerv Syst. 1999;76(1):45-61. doi:10.1016/s0165-1838(99)00008-9
Nurgali K, Stebbing MJ, Furness JB. Correlation of electrophysiological and morphological characteristics of enteric neurons in the mouse colon. J Comp Neurol. 2004;468(1):112-124. doi:10.1002/cne.10948
Bornstein JC, Furness JB, Kunze WA. Electrophysiological characterization of myenteric neurons: how do classification schemes relate? J Auton Nerv Syst. 1994;48(1):1-15. doi:10.1016/0165-1838(94)90155-4
Schutte IW, Kroese AB, Akkermans LM. Somal size and location within the ganglia for electrophysiologically identified myenteric neurons of the Guinea pig ileum. J Comp Neurol. 1995;355(4):563-572. doi:10.1002/cne.903550406
Clerc N, Furness JB, Bornstein JC, Kunze WA. Correlation of electrophysiological and morphological characteristics of myenteric neurons of the duodenum in the Guinea-pig. Neuroscience. 1998;82(3):899-914. doi:10.1016/s0306-4522(97)00318-7
Cornelissen W, De Laet A, Kroese AB, Van Bogaert PP, Scheuermann DW, Timmermans JP. Electrophysiological features of morphological Dogiel type II neurons in the myenteric plexus of pig small intestine. J Neurophysiol. 2000;84(1):102-111. doi:10.1152/jn.2000.84.1.102
Cornelissen W, de Laet A, Kroese AB, van Bogaert PP, Scheuermann DW, Timmermans JP. Excitatory synaptic inputs on myenteric Dogiel type II neurones of the pig ileum. J Comp Neurol. 2001;432(2):137-154. doi:10.1002/cne.1093
Shuttleworth CW, Smith TK. Action potential-dependent calcium transients in myenteric S neurons of the Guinea-pig ileum. Neuroscience. 1999;92(2):751-762. doi:10.1016/s0306-4522(99)00012-3
Smith TK, Burke EP, Shuttleworth CW. Topographical and electrophysiological characteristics of highly excitable S neurones in the myenteric plexus of the Guinea-pig ileum. J Physiol (Lond). 1999;517(Pt 3):817-830.
Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature. 1990;347(6295):768-770. doi:10.1038/347768a0
Costa M, Furness JB, Pompolo S, et al. Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the Guinea-pig small intestine. Neurosci Lett. 1992;148(1-2):121-125. doi:10.1016/0304-3940(92)90819-s
Nichols K, Staines W, Krantis A. Neural sites of the human colon colocalize nitric oxide synthase-related NADPH diaphorase activity and neuropeptide Y. Gastroenterology. 1994;107(4):968-975. doi:10.1016/0016-5085(94)90220-8
Hata F, Ishii T, Kanada A, et al. Essential role of nitric oxide in descending inhibition in the rat proximal colon. Biochem Biophys Res Commun. 1990;172(3):1400-1406. doi:10.1016/0006-291x(90)91605-r
Humenick A, Chen BN, Wattchow DA, et al. Characterization of putative interneurons in the myenteric plexus of human colon. Neurogastroenterol Motil. 2021;33(1):e13964. doi:10.1111/nmo.13964
Spencer NJ, Bywater RA, Taylor GS. Disinhibition during myoelectric complexes in the mouse colon. J Auton Nerv Syst. 1998;71(1):37-47. doi:10.1016/s0165-1838(98)00063-0
Takahashi T. Pathophysiological significance of neuronal nitric oxide synthase in the gastrointestinal tract. J Gastroenterol. 2003;38(5):421-430. doi:10.1007/s00535-003-1094-y
Wood JD. Electrical activity of the intestine of mice with hereditary megacolon and absence of enteric ganglion cells. Am J Dig Dis. 1973;18(6):477-488. doi:10.1007/BF01076598
Hillsley K, Jennings LJ, Mawe GM. Neural control of the gallbladder: an intracellular study of human gallbladder neurons. Digestion. 1998;59(2):125-129. doi:10.1159/000007476
Hanani M, Ermilov LG, Schmalz PF, Louzon V, Miller SM, Szurszewski JH. The three-dimensional structure of myenteric neurons in the Guinea-pig ileum. J Auton Nerv Syst. 1998;71(1):1-9. doi:10.1016/s0165-1838(98)00054-x
Gibbins IL, Morris JL. Structure of peripheral synapses: autonomic ganglia. Cell Tissue Res. 2006;326(2):205-220. doi:10.1007/s00441-006-0233-1