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Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

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Abstract

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2–4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 μm2 for myenteric neurons, 750 μm2 for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons.

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Reference

  • Amara SG, Jonas V, Rosenfeld MG, Ong ES, Evans RM (1982) Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298:240–244

    Article  PubMed  CAS  Google Scholar 

  • Anlauf M, Schafer MK, Eiden L, Weihe E (2003) Chemical coding of the human gastrointestinal nervous system: cholinergic, VIPergic, and catecholaminergic phenotypes. J Comp Neurol 459:90–111

    Article  PubMed  CAS  Google Scholar 

  • Barada KA, Saade NE, Atweh SF, Khoury CI, Nassar CF (2000) Calcitonin gene-related peptide regulates amino acid absorption across rat jejunum. Regul Pept 90:39–45

    Article  PubMed  CAS  Google Scholar 

  • Bartho L, Lembeck F, Holzer P (1987) Calcitonin gene-related peptide is a potent relaxant of intestinal muscle. Eur J Pharmacol 135:449–451

    Article  PubMed  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 

  • Brain SD, Williams TJ (1985) Inflammatory oedema induced by synergism between calcitonin gene-related peptide (CGRP) and mediators of increased vascular permeability. Br J Pharmacol 86:855–860

    PubMed  CAS  Google Scholar 

  • Brehmer A, Schrödl F, Neuhuber W (2002) Correlated morphological and chemical phenotyping in myenteric type V neurons of porcine ileum. J Comp Neurol 453:1–9

    Article  PubMed  CAS  Google Scholar 

  • Brehmer A, Croner R, Dimmler A, Papadopoulos T, Schrödl F, Neuhuber W (2004) Immunohistochemical characterization of putative primary afferent (sensory) myenteric neurons in human small intestine. Autonomic Neurosci 112:49–59

    Article  CAS  Google Scholar 

  • Chiocchetti R, Clavenzani P, Barazzoni AM, Grandis A, Bombardi C, Lalatta Costerbosa G, Petrosino G, Bortolami R (2003) Viscerotopic representation of the subdiaphragmatic tracts of the digestive apparatus within the vagus complex in the sheep. Brain Res 961:32–44

    Article  PubMed  CAS  Google Scholar 

  • Chiocchetti R, Grandis A, Bombardi C, Clavenzani P, Lalatta Costerbosa G, 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, Clavenzani P, Mazzoni M, Albanese V, Di Guardo G, De Grossa L, Bortolami R, Lalatta Costerbosa G (2005) The sheep enteric nervous system after scrapie (PrPsc) infection. Autonomic Neurosci 119:144–145

    Google Scholar 

  • Clague JR, Sternini C, Brecha NC (1985) Localization of calcitonin gene-related peptide-like immunoreactivity in neurons of the rat gastrointestinal tract. Neurosci Lett 56:63–68

    Article  PubMed  CAS  Google Scholar 

  • Cottrell DF, Greenhorn JG (1987) The vagal and spinal innervation of the gastro-duodenal junction of sheep. Q J Exp Physiol 72:513–524

    PubMed  CAS  Google Scholar 

  • Eysselein VE, Reinshagen M, Cominelli F, Sternini C, Davis W, Patel A, Nast CC, Bernstein D, Anderson K, Khan H, Snape WJ (1991) Calcitonin gene-related peptide and substance P decrease in the rabbit colon during colitis. A time study. Gastroenterology 101:1211–1219

    PubMed  CAS  Google Scholar 

  • Feher E, Burnstock G, Varndell IM, Polak JM (1986) Calcitonin gene-related peptide-immunoreactive nerve fibres in the small intestine of the guinea-pig: electron-microscopic immunocytochemistry. Cell Tissue Res 245:353–358

    Article  PubMed  CAS  Google Scholar 

  • Franco-Cereceda A, Henke H, Lundberg JM, Petermann JB, Hokfelt T, Fischer JA (1987) Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin. Peptides 8:399–410

    Article  PubMed  CAS  Google Scholar 

  • Freeman JG, Ryan JJ, Shelburne CP, Bailey DP, Bouton AL, Narasimhachari N, Domen J, Siméon N, Couderc F, Stewart JK (2001) Catecholamines in murine bone marrow derived mast cells. J Neuroimmunol 119:231–238

    Article  PubMed  CAS  Google Scholar 

  • Furness JB, Clerc N, Vogalis F, Stebbing MJ (2003) The enteric nervous system and its extrinsic connections. In: Yamada T (ed) Textbook of gastroenterology. Lippincott, Williams & Wilkins, Philadelphia, pp 12–34

    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 

  • Gabella G, Trigg P (1984) Size of neurons and glial cells in the enteric ganglia of mice, guinea-pigs, rabbits and sheep. J Neurocytol 13:49–71

    Article  PubMed  CAS  Google Scholar 

  • Gates TS, Zimmerman RP, Mantyh CR, Vigna SR, Mantyh PW (1989) Calcitonin gene-related peptide-alpha receptor binding sites in the gastrointestinal tract. Neuroscience 31:757–770

    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 

  • Goehler LE, Sternini C, Brecha NC (1988) Calcitonin gene-related peptide immunoreactivity in the biliary pathway and liver of the guinea-pig: distribution and colocalization with substance P. Cell Tissue Res 253:145–150

    Article  PubMed  CAS  Google Scholar 

  • Green T, Dockray GJ (1987) Calcitonin gene-related peptide and substance P in afferents to the upper gastrointestinal tract in the rat. Neurosci Lett 76:151–156

    Article  PubMed  CAS  Google Scholar 

  • Green T, Dockray GJ (1988) Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig. Neuroscience 25:181–193

    Article  PubMed  CAS  Google Scholar 

  • Grider JR (1994) CGRP as a transmitter in the sensory pathway mediating peristaltic reflex. Am J Physiol 266:G1139–G1145

    PubMed  CAS  Google Scholar 

  • Grider JR, Kuemmerle JF, Jin JG (1996) 5-HT released by mucosal stimuli initiates peristalsis by activating 5-HT4/5-HT1p receptors on sensory CGRP neurons. Am J Physiol 270:G778–G782

    PubMed  CAS  Google Scholar 

  • Ichikawa S, Shiozawa M, Iwanaga T, Uchino S (1991) Immunohistochemical demonstration of peptidergic nerve fibers associated with the central lacteal lymphatics in the duodenal villi of dogs. Arch Histol Cytol 54:241–248

    Article  PubMed  CAS  Google Scholar 

  • Ichikawa S, Sreedharan SP, Goetzl EJ, Owen RL (1994) Immunohistochemical localization of peptidergic nerve fibers and neuropeptide receptors in Peyer's patches of the cat ileum. Regul Pept 54:385–395

    Article  PubMed  CAS  Google Scholar 

  • Kawasaki H (2002) Regulation of vascular function by perivascular calcitonin gene-related peptide-containing nerves. Jpn J Pharmacol 88:39–43

    Article  PubMed  CAS  Google Scholar 

  • Keast JR, Furness JB, Costa M (1985) Distribution of certain peptide-containing nerve fibres and endocrine cells in the gastrointestinal mucosa in five mammalian species. J Comp Neurol 236:403–422

    Article  PubMed  CAS  Google Scholar 

  • Kirchgessner AL, Tamir H, Gershon MD (1992) Identification and stimulation by serotonin of intrinsic sensory neurons of the submucosal plexus of the guinea pig gut: activity-induced expression of Fos immunoreactivity. J Neurosci 12:235–248

    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 

  • 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 

  • Neild TO, Shen KZ, Surprenant A (1990) Vasodilation of arterioles by acetylcholine released from single neurones in the guinea-pig submucosal plexus. J Physiol (Lond) 420:247–265

    CAS  Google Scholar 

  • Palmer JM, Schemann M, Tamura K, Wood JD (1986) Calcitonin gene-related peptide excites myenteric neurons. Eur J Pharmacol 132:163–170

    Article  PubMed  CAS  Google Scholar 

  • Pan H, Gershon MD (2000) Activation of intrinsic afferent pathways in submucosal ganglia of the guinea pig small intestine. J Neurosci 20:3295–3309

    PubMed  CAS  Google Scholar 

  • Pfannkuche H, Schemann M, Gäbel G (2002) Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones. Cell Tissue Res 309:347–354

    Article  PubMed  CAS  Google Scholar 

  • Rasmussen TN, Gregersen H, Harling H, Holst JJ (1992) Calcitonin gene-related peptide: effect on contractile activity and luminal cross-sectional area in the isolated, perfused porcine ileum. Scand J Gastroenterol 27:787–792

    Article  PubMed  CAS  Google Scholar 

  • Rasmussen TN, Schmidt P, Poulsen SS, Holst JJ (2001) Localisation and neural control of the release of calcitonin gene-related peptide (CGRP) from the isolated perfused porcine ileum. Regul Pept 98:137–143

    Article  PubMed  CAS  Google Scholar 

  • Rosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vale WW, Evans RM (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304:129–135

    Article  PubMed  CAS  Google Scholar 

  • Scheuermann DW, Stach W, De Groodt-Lasseel MHA, Timmermans J-P (1987) Calcitonin gene-related peptide in morphologically well-defined type II neurons of the enteric nervous system in the porcine small intestine. Acta Anat 129:325–328

    PubMed  CAS  Google Scholar 

  • Scheuermann DW, Krammer H-J, Timmermans J-P, 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 

  • Sternini C (1991) Tachykinin and calcitonin gene-related peptide immunoreactivities and mRNAs in the mammalian enteric nervous system and sensory ganglia. Adv Exp Med Biol 298:39–51

    PubMed  CAS  Google Scholar 

  • Sternini C (1992) Enteric and visceral afferent CGRP neurons. Targets of innervation and differential expression patterns. Ann NY Acad Sci 657:170–186

    Article  PubMed  CAS  Google Scholar 

  • Sternini C, Anderson K (1992) Calcitonin gene-related peptide-containing neurons supplying the rat digestive system: differential distribution and expression pattern. Somatosens Motor Res 9:45–59

    Article  CAS  Google Scholar 

  • Sternini C, Reeve JR, Brecha NC (1987) Distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal and capsaicin-treated rats. Gastroenterology 93:852–862

    PubMed  CAS  Google Scholar 

  • Su HC, Bishop AE, Power RF, Hamada Y, Polak JM (1987) Dual intrinsic and extrinsic origins of CGRP- and NPY-immunoreactive nerves of rat gut and pancreas. J Neuroscience 7:2674–2687

    PubMed  CAS  Google Scholar 

  • Taché Y (1992) Inhibition of gastric acid secretion and ulcers by calcitonin gene-related peptide. Ann NY Acad Sci 657:240–247

    Article  PubMed  Google Scholar 

  • Timmermans J-P, Scheuermann DW, Barbiers M, Adriaensen D, Stach W, Van Hee R, De Groodt-Lasseel MHA (1992) Calcitonin gene-related peptide-like immunoreactivity in the human small intestine. Acta Anat 143:48–53

    PubMed  CAS  Google Scholar 

  • Vanner S (1994) Co-release of neuropeptides from capsaicin-sensitive afferents dilates submucosal arterioles in the guinea-pig ileum. Am J Physiol 267:G650–G655

    PubMed  CAS  Google Scholar 

  • Vanner S, Surprenant A (1996) Neuronal reflexes controlling intestinal microcirculation. Am J Physiol 271:G223–G230

    PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Valeria Albanese and Beatrice Travostini for their invaluable assistance and advice in the preparation of tissue for immunohistochemistry.

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Authors and Affiliations

  1. Department of Veterinary Morphophysiology and Animal Productions, University of Bologna, 40064, Bologna, Ozzano Emilia, Italy

    Roberto Chiocchetti, Annamaria Grandis, Cristiano Bombardi, Maria L. Lucchi, Davide Tadini Dal Lago & Ruggero Bortolami

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

    John B. Furness

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  1. Roberto Chiocchetti
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  2. Annamaria Grandis
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  3. Cristiano Bombardi
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  4. Maria L. Lucchi
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  6. Ruggero Bortolami
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  7. John B. Furness
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Correspondence to Roberto Chiocchetti.

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This work was supported by grants from the Ricerca Fondamentale Orientata (RFO) and Fondazione Del Monte di Bo e Ra.

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Chiocchetti, R., Grandis, A., Bombardi, C. et al. 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 (2006). https://doi.org/10.1007/s00441-005-0075-2

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