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Immunohistochemical analysis of neuron types in the mouse small intestine

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Abstract

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/± TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

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References

  • Abdu F, Hicks GA, Hennig G, Allen JP, Grundy D (2002) Somatostatin sst2 receptors inhibit peristalsis in the rat and mouse jejunum. Am J Physiol 282:G624–G633

    CAS  Google Scholar 

  • Barbiers M, Timmermans JP, Adriaensen D, De Groodt Lasseel MHA, Scheuermann DW (1995) Projections of neurochemically specified neurons in the porcine colon. Histochemistry 103:115–126

    Article  PubMed  CAS  Google Scholar 

  • Bian X, Ren J, DeVries M, Schnegelsberg B, Cockayne DA, Ford APDW, Galligan JJ (2003) Peristalsis is impaired in the small intestine of mice lacking the P2X3 subunit. J Physiol (Lond) 551:309–322

    Article  CAS  Google Scholar 

  • Bornstein JC, Hendriks R, Furness JB, Trussell DC (1991) Ramifications of the axons of AH-neurons injected with the intracellular marker biocytin in the myenteric plexus of the guinea pig small intestine. J Comp Neurol 314:437–451

    Article  PubMed  CAS  Google Scholar 

  • Brehmer A (2006) Structure of enteric neurons. Adv Anat 186:1–95

    CAS  Google Scholar 

  • Brehmer A (2007) The value of neurofilament-immunohistochemistry for identifying enteric neuron types—special reference to intrinsic primary afferent (sensory) neurons. In: Arlen RK (ed) New research on neurofilament proteins. Nova Science, New York, pp 99–114

    Google Scholar 

  • Brehmer A, Stach W, Krammer HJ, Neuhuber W (1998) Distribution, morphology and projections of nitrergic and non-nitrergic submucosal neurons in the pig small intestine. Histochem Cell Biol 109:87–94

    Article  PubMed  CAS  Google Scholar 

  • Brehmer A, Schrödl F, Neuhuber W (2002) Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum. Histochem Cell Biol 117:257–263

    Article  PubMed  CAS  Google Scholar 

  • Brookes SJH (2001) Classes of enteric nerve cells in the guinea-pig small intestine. Anat Rec 262:58–70

    Article  PubMed  CAS  Google Scholar 

  • Brookes SJH, Steele PA, Costa M (1991) Identification and immunohistochemistry of cholinergic and non-cholinergic circular muscle motor neurons in the guinea-pig small intestine. Neuroscience 42:863–878

    Article  PubMed  CAS  Google Scholar 

  • Cajal SRY (1911) Histologie du système nerveux de l’homme et des vertébrés. Maloine, Paris

    Google Scholar 

  • Chiocchetti R, Grandis A, Bombardi C, Clavenzani P, Costerbosa GL, Lucchi ML, Furness JB (2004) Characterisation of neurons expressing calbindin immunoreactivity in the ileum of the unweaned and mature sheep. Cell Tissue Res 318:289–303

    Article  PubMed  CAS  Google Scholar 

  • Chiocchetti R, Grandis A, Bombardi C, Lucchi ML, Dal Largo DT, Bortolami R, Furness JB (2006) Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation. Cell Tissue Res 323:183–196

    Article  PubMed  CAS  Google Scholar 

  • Clarke CM, Plata C, Cole B, Tsuchiya K, La Spada AR, Kapur RP (2007) Visceral neuropathy and intestinal pseudo-obstruction in a murine model of a nuclear inclusion disease. Gastroenterology 133:1971–1978

    Article  PubMed  CAS  Google Scholar 

  • Costa M, Furness JB, Pompolo S, Brookes SJH, Bornstein JC, Bredt DS, Snyder SH (1992) Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine. Neurosci Lett 148:121–125

    Article  PubMed  CAS  Google Scholar 

  • Dahl D, Bignami A (1977) Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve. J Comp Neurol 176:645–657

    Article  PubMed  CAS  Google Scholar 

  • De Jonge F, Van Nassauw L, Adriaensen D, Van Meir F, Miller HRP, Van Marck E, Timmermans J-P (2003a) Effect of intestinal inflammation on capsaicin-sensitive afferents in the ileum of Schistosoma mansoni-infected mice. Histochem Cell Biol 119:477–484

    Article  PubMed  Google Scholar 

  • De Jonge F, Van Nassauw L, De Man JG, De Winter BY, Van Meir F, Depoortere I, Peeters TL, Pelckmans PA, Van Marck E, Timmermans J-P (2003b) Effects of Schistosoma mansoni infection on somatostatin and somatostatin receptor 2A expression in mouse ileum. Neurogastroenterol Motil 15:149–159

    Article  PubMed  Google Scholar 

  • Dogiel AS (1899) Über den Bau der Ganglien in den Geflechten des Darmes und der Gallenblase des Menschen und der Säugetiere. Arch Anat Physiol Leipzig Anat Abt Jg 1899:130–158

    Google Scholar 

  • Fairman CL, Clagett Dame M, Lennon VA, Epstein ML (1995) Appearance of neurons in the developing chick gut. Dev Dyn 204:192–201

    PubMed  CAS  Google Scholar 

  • Furness JB (2000) Types of neurons in the enteric nervous system. J Auton Nerv Syst 81:87–96

    Article  PubMed  CAS  Google Scholar 

  • Furness JB (2006) The enteric nervous system. Blackwell, Oxford

    Google Scholar 

  • Furness JB, Costa M (1971) Morphology and distribution of intrinsic adrenergic neurones in the proximal colon of the guinea-pig. Z Zellforsch 120:346–363

    Article  Google Scholar 

  • Furness JB, Costa M (1979) Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons. Neurosci Lett 15:199–204

    Article  PubMed  CAS  Google Scholar 

  • Furness JB, Costa M (1982) Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: their projections in the guinea-pig small intestine. Neuroscience 7:341–349

    Article  PubMed  CAS  Google Scholar 

  • Furness JB, Lloyd KCK, Sternini C, Walsh JH (1990a) Projections of substance P, vasoactive intestinal peptide and tyrosine hydroxylase immunoreactive nerve fibres in the canine intestine, with special reference to the innervation of the circular muscle. Arch Histol Cytol 53:129–140

    Article  PubMed  CAS  Google Scholar 

  • Furness JB, Trussell DC, Pompolo S, Bornstein JC, Smith TK (1990b) Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections. Cell Tissue Res 260:261–272

    Article  PubMed  CAS  Google Scholar 

  • Furness JB, Clerc N, Lomax AEG, Bornstein JC, Kunze WAA (2000) Shapes and projections of tertiary plexus neurons of the guinea-pig small intestine. Cell Tissue Res 300:383–387

    Article  PubMed  CAS  Google Scholar 

  • Furness JB, Alex G, Clark MJ, Lal VV (2003) Morphologies and projections of defined classes of neurons in the submucosa of the guinea-pig small intestine. Anat Rec 272A:475–483

    Article  Google Scholar 

  • Furness JB, Jones C, Nurgali K, Clerc N (2004a) Intrinsic primary afferent neurons and nerve circuits within the intestine. Prog Neurobiol 72:143–164

    Article  PubMed  CAS  Google Scholar 

  • Furness JB, Robbins HL, Xiao J, Stebbing MJ, Nurgali K (2004b) Projections and chemistry of Dogiel type II neurons in the mouse colon. Cell Tissue Res 317:1–12

    Article  PubMed  CAS  Google Scholar 

  • Gibbins IL, Furness JB, Costa M, MacIntyre I, Hillyard CJ, Girgis S (1985) Co-localization of calcitonin gene related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea-pigs. Neurosci Lett 57:125–130

    Article  PubMed  CAS  Google Scholar 

  • Grider JR (2003) Neurotransmitters mediating the intestinal peristaltic reflex in the mouse. J Pharmacol Exp Ther 307:460–467

    Article  PubMed  CAS  Google Scholar 

  • Heanue TA, Pachnis V (2007) Enteric nervous system development and Hirschsprung’s disease: advances in genetic and stem cell studies. Nat Rev Neurosci 8:466–479

    Article  PubMed  CAS  Google Scholar 

  • Hens J, Schrödl F, Brehmer A, Adriaensen D, Neuhuber W, Scheuermann DW, Schemann M, Timmermans J-P (2000) Mucosal projections of enteric neurons in the porcine small intestine. J Comp Neurol 421:426–436

    Article  Google Scholar 

  • Hens J, Vanderwinden J-M, De Laet MH, Scheuermann DW, Timmermans J-P (2001) Morphological and neurochemical identification of enteric neurones with mucosal projections in the human small intestine. J Neurochem 76:464–471

    Article  PubMed  CAS  Google Scholar 

  • Kunze WAA, Clerc N, Furness JB, Gola M (2000) The soma and neurites of primary afferent neurons in the guinea-pig intestine respond differentially to deformation. J Physiol (Lond) 526:375–385

    Article  CAS  Google Scholar 

  • Li ZS, Furness JB (1998) Immunohistochemical localization of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine. Cell Tissue Res 294:35–43

    Article  PubMed  CAS  Google Scholar 

  • Li ZS, Pham TD, Tamir H, Chen JJ, Gershon MD (2004) Enteric dopaminergic neurons: definition, developmental lineage, and effects of extrinsic denervation. J Neurosci 24:1330–1339

    Article  PubMed  CAS  Google Scholar 

  • Lyster DJK, Bywater RAR, Taylor GS (1995) Neurogenic control of myoelectric complexes in the mouse isolated colon. Gastroenterology 108:1371–1378

    Article  PubMed  CAS  Google Scholar 

  • Mao Y, Wang B, Kunze W (2006) Characterization of myenteric sensory neurons in the mouse small intestine. J Neurophysiol 96:998–1010

    Article  PubMed  Google Scholar 

  • Mashimo H, Kjellin A, Goyal RK (2000) Gastric stasis in neuronal nitric oxide synthase-deficient knockout mice. Gastroenterology 119:766–773

    Article  PubMed  CAS  Google Scholar 

  • Messenger JP, Furness JB (1990) Projections of chemically-specified neurons in the guinea-pig colon. Arch Histol Cytol 53:467–495

    Article  PubMed  CAS  Google Scholar 

  • Morris JL, Gibbins IL, Campbell G, Murphy R, Furness JB, Costa M (1986) Innervation of the large arteries and heart of the toad Bufo marinus by adrenergic and peptide-containing neurons. Cell Tissue Res 243:171–184

    Article  PubMed  CAS  Google Scholar 

  • Newgreen D, Young HM (2002a) Enteric nervous system: development and developmental disturbances—part 1. Pediatr Dev Pathol 5:224–247

    PubMed  CAS  Google Scholar 

  • Newgreen D, Young HM (2002b) Enteric nervous system: development and developmental disturbances—part 2. Pediatr Dev Pathol 5:329–349

    Article  PubMed  Google Scholar 

  • Nurgali K, Stebbing MJ, Furness JB (2004) Correlation of electrophysiological and morphological characteristics of enteric neurons in the mouse colon. J Comp Neurol 468:112–124

    Article  PubMed  Google Scholar 

  • Pompolo S, Furness JB (1998) Quantitative analysis of inputs to somatostatin immunoreactive descending interneurons in the myenteric plexus of the guinea-pig small intestine. Cell Tissue Res 294:219–226

    Article  PubMed  CAS  Google Scholar 

  • Portbury AL, Pompolo S, Furness JB, Stebbing MJ, Kunze WAA, Bornstein JC, Hughes S (1995) Cholinergic, somatostatin-immunoreactive interneurons in the guinea pig intestine: morphology, ultrastructure, connections and projections. J Anat 187:303–321

    PubMed  Google Scholar 

  • Porter AJ, Wattchow DA, Brookes SJH, Costa M (1997) The neurochemical coding and projections of circular muscle motor neurons in the human colon. Gastroenterology 113:1916–1923

    Article  PubMed  CAS  Google Scholar 

  • Ren J, Bian X, DeVries M, Schnegelsberg B, Cockayne DA, Ford APDW, Galligan JJ (2003) P2X2 subunits contribute to fast synaptic excitation in myenteric neurons of the mouse small intestine. J Physiol (Lond) 552:809–821

    Article  CAS  Google Scholar 

  • Roberts RR, Bornstein JC, Bergner AJ, Young HM (2008) Disturbances of colonic motility in mouse models of Hirschsprung’s disease. Am J Gastroenterol 294:G996–G1008

    CAS  Google Scholar 

  • Sang Q, Young HM (1996) Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse. Cell Tissue Res 284:39–53

    Article  PubMed  CAS  Google Scholar 

  • Sang Q, Young HM (1998) The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse. Anat Rec 251:185–199

    Article  PubMed  CAS  Google Scholar 

  • Sang Q, Williamson S, Young HM (1997) Projections of chemically identified myenteric neurons of the small and large intestine of the mouse. J Anat 190:209–222

    Article  PubMed  CAS  Google Scholar 

  • Scheuermann DW, Krammer HJ, Timmermans JP, Stach W, Adriaensen D (1991) Fine structure of morphologically well-defined type II neurons in the enteric nervous system of the porcine small intestine revealed by immunoreactivity for calcitonin gene-related peptide. Acta Anat 142:236–241

    Article  PubMed  CAS  Google Scholar 

  • Song ZM, Brookes SJH, Costa M (1994) All calbindin-immunoreactive myenteric neurons project to the mucosa of the guinea-pig small intestine. Neurosci Lett 180:219–222

    Article  PubMed  CAS  Google Scholar 

  • Stach W (1989) A revised morphological classification of neurons in the enteric nervous system. In: Singer MV, Goebell H (eds) Nerves and the gastrointestinal tract. MTP, Lancaster, pp 29–45

    Google Scholar 

  • Timmermans J-P, Scheuermann DW, Stach W, Barbiers M, Adriaensen D, Krammer HJ, De Groodt Lasseel MHA (1991) Projections of serotonin-containing neurons in the enteric nerve plexus, an experimental study in the porcine small intestine. Verh Anat Ges 85:89–90

    Google Scholar 

  • Timmermans J-P, Barbiers M, Scheuermann DW, Stach W, Adriaensen D, Mayer B, De Groodt Lasseel MHA (1994) Distribution pattern, neurochemical features and projections of nitrergic neurons in the pig small intestine. Ann Anat 176:515–525

    PubMed  CAS  Google Scholar 

  • Timmermans J-P, Adriaensen D, Cornelissen W, Scheuermann DW (1997) Structural organization and neuropeptide distribution in the mammalian enteric nervous system, with special attention to those components involved in mucosal reflexes. Comp Biochem Physiol 118A:331–340

    Article  CAS  Google Scholar 

  • Timmermans J-P, Hens J, Adriaensen D (2001) Outer submucous plexus: an intrinsic nerve network involved in both secretory and motility processes in the intestine of large mammals and humans. Anat Rec 262:71–78

    Article  PubMed  CAS  Google Scholar 

  • Van Nassauw L, Hens J, Bogers J, Van Marck E, Timmermans J-P (2000) Retrograde DiI tracing of enteric neurons projecting to the mucosa in the murine small intestine. In: Krammer H-J, Singer MV (eds) Neurogastroenterology, from the basics to the clinics. Kluwer Academic, Dordrecht, pp 92–95

    Google Scholar 

  • Van Op Den Bosch J, Lantermann K, Torfs P, Van Marck E, Van Nassauw L, Timmermans J-P (2008) Distribution and expression levels of somatostatin and somatostatin receptors in the ileum of normal and acutely Schistosoma mansoni-infected SSTR2 knockout/lacZ knockin mice. Neurogastroenterol Motil 20:798–807

    Article  PubMed  Google Scholar 

  • Ward SM, McLaren GJ, Sanders KM (2006) Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine. J Physiol (Lond) 573:147–159

    Article  CAS  Google Scholar 

  • Wardell CF, Bornstein JC, Furness JB (1994) Projections of 5-hydroxytryptamine-immunoreactive neurons in guinea-pig distal colon. Cell Tissue Res 278:379–387

    Article  PubMed  CAS  Google Scholar 

  • Weidmann S, Schrödl F, Neuhuber W, Brehmer A (2007) Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine. Histochem Cell Biol 128:399–407

    Article  PubMed  CAS  Google Scholar 

  • Weihe E, Tao Cheng JH, Schäfer MKH, Erickson JD, Eiden LE (1996) Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles. Proc Natl Acad Sci USA 93:3547–3552

    Article  PubMed  CAS  Google Scholar 

  • Williamson S, Pompolo S, Furness JB (1996) GABA and nitric oxide synthase immunoreactivities are colocalized in a subset of inhibitory motor neurons of the guinea-pig small intestine. Cell Tissue Res 284:29–37

    Article  PubMed  CAS  Google Scholar 

  • Young HM, Ciampoli D (1998) Transient expression of neuronal nitric oxide synthase by neurons of the submucous plexus of the mouse small intestine. Cell Tissue Res 291:395–401

    Article  PubMed  CAS  Google Scholar 

  • Zhao A, Urban JFJ, Morimoto M, Elfrey JE, Madden KB, Finkelman FD, Shea-Donohue T (2006) Contribution of 5-HT2A receptor in nematode infection-induced murine intestinal smooth muscle hypercontractility. Gastroenterology 131:568–578

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Dr. Heather Young for her valuable comments on the manuscript.

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Authors and Affiliations

  1. Department of Anatomy & Cell Biology and Centre for Neuroscience, University of Melbourne, Parkville, Victoria, 3010, Australia

    Zheng-Dong Qu, Michelle Thacker, Patricia Castelucci, Mária Bagyánszki & John B. Furness

  2. Department of Anatomy, University of São Paulo, São Paulo, Brazil

    Patricia Castelucci

  3. Department of Physiology, Anatomy and Neuroscience, University of Szeged, H-6726, Szeged, Hungary

    Mária Bagyánszki

  4. Department of Anatomy, University of Wisconsin Medical School, Madison, WI 53706, USA

    Miles L. Epstein

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  1. Zheng-Dong Qu
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  2. Michelle Thacker
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  3. Patricia Castelucci
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  4. Mária Bagyánszki
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  5. Miles L. Epstein
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  6. John B. Furness
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Correspondence to John B. Furness.

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This work was supported by a grant from the National Health and Medical Council of Australia (grant number 400020) and a FAPESP Fellowship (06/57532–0) from the Fundação de Amparo a Pesquisa do Estado de São Paulo.

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Qu, ZD., Thacker, M., Castelucci, P. et al. Immunohistochemical analysis of neuron types in the mouse small intestine. Cell Tissue Res 334, 147–161 (2008). https://doi.org/10.1007/s00441-008-0684-7

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