Skip to main content

Excitatory and inhibitory enteric innervation of horse lower esophageal sphincter

Histochemistry and Cell Biology volume 143, pages 625–635 (2015)Cite this article

Abstract

The lower esophageal sphincter (LES) is a specialized, thickened muscle region with a high resting tone mediated by myogenic and neurogenic mechanisms. During swallowing or belching, the LES undergoes strong inhibitory innervation. In the horse, the LES seems to be organized as a “one-way” structure, enabling only the oral-anal progression of food. We characterized the esophageal and gastric pericardial inhibitory and excitatory intramural neurons immunoreactive (IR) for the enzymes neuronal nitric oxide synthase (nNOS) and choline acetyltransferase. Large percentages of myenteric plexus (MP) and submucosal (SMP) plexus nNOS-IR neurons were observed in the esophagus (72 ± 9 and 69 ± 8 %, respectively) and stomach (57 ± 17 and 45 ± 3 %, respectively). In the esophagus, cholinergic MP and SMP neurons were 29 ± 14 and 65 ± 24 vs. 36 ± 8 and 38 ± 20 % in the stomach, respectively. The high percentage of nitrergic inhibitory motor neurons observed in the caudal esophagus reinforces the role of the enteric nervous system in the horse LES relaxation. These findings might allow an evaluation of whether selective groups of enteric neurons are involved in horse neurological disorders such as megaesophagus, equine dysautonomia, and white lethal foal syndrome.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Barone R (1981) Anatomia comparata dei mammiferi domestici, 1st edn. Edagricole, Bologna

    Google Scholar 

  • Beck M, Schlabrakowski A, Schrodl F, Neuhuber W, Brehmer A (2009) ChAT and NOS in human myenteric neurons: co-existence and co-absence. Cell Tissue Res 338(1):37–51. doi:10.1007/s00441-009-0852-4

    Article  CAS  PubMed  Google Scholar 

  • Boeckxstaens GE (2005) The lower oesophageal sphincter. Neurogastroenterol Motil 17(Suppl 1):13–21. doi:10.1111/j.1365-2982.2005.00661.x

    Article  PubMed  Google Scholar 

  • Branchek TA, Gershon MD (1989) Time course of expression of neuropeptide Y, calcitonin gene-related peptide, and NADPH diaphorase activity in neurons of the developing murine bowel and the appearance of 5-hydroxytryptamine in mucosal enterochromaffin cells. J Comp Neurol 285(2):262–273. doi:10.1002/cne.902850208

    Article  CAS  PubMed  Google Scholar 

  • Brehmer A, Schrodl F, Neuhuber W, Tooyama I, Kimura H (2004) Co-expression pattern of neuronal nitric oxide synthase and two variants of choline acetyltransferase in myenteric neurons of porcine ileum. J Chem Neuroanat 27(1):33–41

    Article  CAS  PubMed  Google Scholar 

  • Brehmer A, Rupprecht H, Neuhuber W (2010) Two submucosal nerve plexus in human intestines. Histochem Cell Biol 133(2):149–161. doi:10.1007/s00418-009-0657-2

    Article  CAS  PubMed  Google Scholar 

  • Brookes SJ, Chen BN, Hodgson WM, Costa M (1996) Characterization of excitatory and inhibitory motor neurons to the guinea pig lower esophageal sphincter. Gastroenterology 111(1):108–117

    Article  CAS  PubMed  Google Scholar 

  • Bueno L, Ruckebusch Y (1979) Perinatal development of intestinal myoelectrical activity in dogs and sheep. Am J Physiol 237(1):E61–E67

    CAS  PubMed  Google Scholar 

  • Chiocchetti R, Bombardi C, Mongardi-Fantaguzzi C, Venturelli E, Russo D, Spadari A, Montoneri C, Romagnoli N, Grandis A (2009a) Intrinsic innervation of the horse ileum. Res Vet Sci 87(2):177–185

    Article  PubMed  Google Scholar 

  • Chiocchetti R, Bombardi C, Mongardi Fantaguzzi C, Russo D, Venturelli E, Montoneri C, Spadari A, Romagnoli N, Grandis A (2009b) Intrinsic innervation of the ileocaecal junction in the horse: preliminary study. Equine Vet J 41(8):759–764

    Article  CAS  PubMed  Google Scholar 

  • Christensen HM (1973) Government and the future of dentistry. Alumni bull 77–83

  • Clark ES, Morris DD, Whitlock RH (1987) Esophageal manometry in horses, cows, and sheep during deglutition. Am J Vet Res 48(4):547–551

    CAS  PubMed  Google Scholar 

  • Clerc N (1983) Afferent innervation of the lower oesophageal sphincter of the cat. An HRP study. J Auton Nerv Syst 9(4):623–636

    Article  CAS  PubMed  Google Scholar 

  • Collman PI, Tremblay L, Diamant NE (1992) The distribution of spinal and vagal sensory neurons that innervate the esophagus of the cat. Gastroenterology 103(3):817–822

    CAS  PubMed  Google Scholar 

  • Collman PI, Tremblay L, Diamant NE (1993) The central vagal efferent supply to the esophagus and lower esophageal sphincter of the cat. Gastroenterology 104(5):1430–1438

    CAS  PubMed  Google Scholar 

  • Conklin JL (1998) Nitric oxide: a mediator of esophageal motor function. J Lab Clin Med 131(1):10–20

    Article  CAS  PubMed  Google Scholar 

  • Cottrell DF, McGorum BC, Pearson GT (1999) The neurology and enterology of equine grass sickness: a review of basic mechanisms. Neurogastroenterol Motil 11(2):79–92

    Article  CAS  PubMed  Google Scholar 

  • Delmann HD (2000) Anatomia microscopica veterinaria, 1st edn. Ambrosiana, Milano

    Google Scholar 

  • Dong H, Jiang Y, Srinivasan S, Mittal RK (2013) Morphological, immunocytochemical, and functional characterization of esophageal enteric neurons in primary culture. Am J Physiol Gastrointest Liver Physiol 305(2):G129–G138. doi:10.1152/ajpgi.00040.2013

    Article  CAS  PubMed  Google Scholar 

  • Fang S, Christensen J (1994) Distribution of NADPH diaphorase in intramural plexuses of cat and opossum esophagus. J Auton Nerv Syst 46(1–2):123–133

    Article  CAS  PubMed  Google Scholar 

  • Farre R, Sifrim D (2008) Regulation of basal tone, relaxation and contraction of the lower oesophageal sphincter. Relevance to drug discovery for oesophageal disorders. Br J Pharmacol 153(5):858–869

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Freytag C, Seeger J, Siegemund T, Grosche J, Grosche A, Freeman DE, Schusser GF, Hartig W (2008) Immunohistochemical characterization and quantitative analysis of neurons in the myenteric plexus of the equine intestine. Brain Res 1244:53–64

    Article  CAS  PubMed  Google Scholar 

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

    Google Scholar 

  • Furness JB (2012) The enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol 9(5):286–294

    Article  CAS  PubMed  Google Scholar 

  • Furness JB, Lloyd KC, Sternini C, Walsh JH (1990) 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(2):129–140

    Article  CAS  PubMed  Google Scholar 

  • Furness JB, Li ZS, Young HM, Forstermann U (1994) Nitric oxide synthase in the enteric nervous system of the guinea-pig: a quantitative description. Cell Tissue Res 277(1):139–149

    Article  CAS  PubMed  Google Scholar 

  • Goyal RK, Chaudhury A (2008) Physiology of normal esophageal motility. J Clin Gastroenterol 42(5):610–619. doi:10.1097/MCG.0b013e31816b444d

    Article  PubMed Central  PubMed  Google Scholar 

  • Goyal RK, Rattan S (1975) Nature of the vagal inhibitory innervation to the lower esophageal sphincter. J Clin Invest 55(5):1119–1126. doi:10.1172/JCI108013

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Grozdanovic Z, Baumgarten HG, Bruning G (1992) Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse. Neuroscience 48(1):225–235

    Article  CAS  PubMed  Google Scholar 

  • Hempfling C, Seibold R, Shiina T, Heimler W, Neuhuber WL, Worl J (2009) Enteric co-innervation of esophageal striated muscle fibers: a phylogenetic study. Auton Neurosci 151(2):135–141

    Article  CAS  PubMed  Google Scholar 

  • Hornby PJ, Abrahams TP (2000) Central control of lower esophageal sphincter relaxation. Am J Med 108(Suppl 4a):90S–98S

    Article  PubMed  Google Scholar 

  • Hultgren BD (1982) Ileocolonic aganglionosis in white progeny of overo spotted horses. J Am Vet Med Assoc 180(3):289–292

    CAS  PubMed  Google Scholar 

  • Kelly KA (1981) Motility of the stomach and gastroduodenal junction. In: Johnson LR (ed) Physiology of the gastrointestinal tract. Raven Press, New York

    Google Scholar 

  • Komine M, Langohr IM, Kiupel M (2014) Megaesophagus in Friesian horses associated with muscular hypertrophy of the caudal esophagus. Vet Pathol 51(5):979–985

  • König HE (2006) Anatomia dei mammiferi domestici, vol 2. Piccin Editore, Padova

    Google Scholar 

  • Kuramoto H, Kadowaki M (2006) Vagus nerve stimulation preferentially induces Fos expression in nitrergic neurons of rat esophagus. Cell Tissue Res 324(3):361–367. doi:10.1007/s00441-005-0124-x

    Article  CAS  PubMed  Google Scholar 

  • Kwiatek MA, Kahrilas PJ (2012) Physiology of the LES. Dis Esophagus 25(4):286–291. doi:10.1111/j.1442-2050.2011.01184.x

    Article  CAS  PubMed  Google Scholar 

  • Lefebvre RA, Smits GJ, Timmermans JP (1995) Study of NO and VIP as non-adrenergic non-cholinergic neurotransmitters in the pig gastric fundus. Br J Pharmacol 116(3):2017–2026

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Long JD, Orlando RC (1999) Esophageal submucosal glands: structure and function. Am J Gastroenterol 94(10):2818–2824

    Article  CAS  PubMed  Google Scholar 

  • Mazzuoli G, Mazzoni M, Albanese V, Clavenzani P, Lalatta-Costerbosa G, Lucchi ML, Furness JB, Chiocchetti R (2007) Morphology and neurochemistry of descending and ascending myenteric plexus neurons of sheep ileum. Anat Rec (Hoboken) 290(12):1480–1491. doi:10.1002/ar.20615

    Article  CAS  Google Scholar 

  • Milla PJ (1993) The ontogeny of intestinal motor activity. In: Kumar D, Wingate D (eds) An illustrated guide to gastrointestinal motility. Churchill Livingstone, Edinburgh, pp 51–63

    Google Scholar 

  • Mittal RK, Bhalla V (2004) Oesophageal motor functions and its disorders. Gut 53(10):1536–1542

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Morikawa S, Komuro T (1998) Distribution of myenteric NO neurons along the guinea-pig esophagus. J Auton Nerv Syst 74(2–3):91–99

    Article  CAS  PubMed  Google Scholar 

  • Muniz E, Lobo Ladd AA, Lobo Ladd FV, da Silva AA, Kmit FV, Borges AS, Teixeira R, da Mota LS, Belli CB, de Zoppa AL, da Silva LC, de Melo MP, Coppi AA (2013) 3-D technology used to accurately understand equine ileocolonic aganglionosis. Cells Tissues Organs 198(2):160–168

    Article  PubMed  Google Scholar 

  • Murphy EM, Defontgalland D, Costa M, Brookes SJ, Wattchow DA (2007) Quantification of subclasses of human colonic myenteric neurons by immunoreactivity to Hu, choline acetyltransferase and nitric oxide synthase. Neurogastroenterol Motil 19(2):126–134. doi:10.1111/j.1365-2982.2006.00843.x

    Article  CAS  PubMed  Google Scholar 

  • Nakajima K, Tooyama I, Yasuhara O, Aimi Y, Kimura H (2000) Immunohistochemical demonstration of choline acetyltransferase of a peripheral type (pChAT) in the enteric nervous system of rats. J Chem Neuroanat 18(1–2):31–40

    Article  CAS  PubMed  Google Scholar 

  • Nickel R (1973) The viscera of domestic mammals. Verlag Paul Parey, New York

    Google Scholar 

  • Niel JP, Gonella J, Roman C (1980) Horseradish peroxidase localization of the cell bodies of the sympathetic and parasympathetic neurones controlling the lower oesophageal sphincter in the cat (author’s transl). J Physiol (Paris) 76(6):591–599

    CAS  Google Scholar 

  • Noorian AR, Taylor GM, Annerino DM, Greene JG (2011) Neurochemical phenotypes of myenteric neurons in the rhesus monkey. J Comp Neurol 519(17):3387–3401. doi:10.1002/cne.22679

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Onori L, Friedmann CA, Frigo GM, Tonini M (1971) Effects of catecholamines, nicotine, acetylcholine and potassium on the mechanical activity of the colonic muscularis mucosae in the cat. Am J Dig Dis 16(8):689–692

    Article  CAS  PubMed  Google Scholar 

  • Pearson GT (1994) Structural organization and neuropeptide distributions in the equine enteric nervous system: an immunohistochemical study using whole-mount preparations from the small intestine. Cell Tissue Res 276(3):523–534

    Article  CAS  PubMed  Google Scholar 

  • Pimont S, Bruley Des Varannes S, Le Neel JC, Aubert P, Galmiche JP, Neunlist M (2003) Neurochemical coding of myenteric neurones in the human gastric fundus. Neurogastroenterol Motil 15(6):655–662

    Article  CAS  PubMed  Google Scholar 

  • Pirie RS, Jago RC, Hudson NP (2014) Equine grass sickness. Equine Vet J. doi:10.1111/evj.12254

    Google Scholar 

  • Rodrigo J, Uttenthal LO, Peinado MA, Esteban FJ, Fernandez AP, Serrano J, Martinez de Velasco J, Santacana M, Bentura ML, Martinez-Murillo R, Pedrosa JA (1998) Distribution of nitric oxide synthase in the esophagus of the cat and monkey. J Auton Nerv Syst 70(3):164–179

    Article  CAS  PubMed  Google Scholar 

  • Rossowski WJ, Tobey NA, Cheng BL, Coy DH, Orlando RC (1996) Vagal stimulation of opossum esophageal submucosal gland bicarbonate secretion. Gastroenterology 110:A244

    Google Scholar 

  • Russo D, Bombardi C, Grandis A, Furness JB, Spadari A, Bernardini C, Chiocchetti R (2010) Sympathetic innervation of the ileocecal junction in horses. J Comp Neurol 518(19):4046–4066. doi:10.1002/cne.22443

    Article  CAS  PubMed  Google Scholar 

  • Sanders KM, Smith TK (1986) Motoneurones of the submucous plexus regulate electrical activity of the circular muscle of canine proximal colon. J Physiol 380:293–310

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sang Q, Young HM (1998) The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus. Brain Res 809(2):253–268

    Article  CAS  PubMed  Google Scholar 

  • Sang Q, Ciampoli D, Greferath U, Sommer L, Young HM (1999) Innervation of the esophagus in mice that lack MASH1. J Comp Neurol 408(1):1–10

    Article  CAS  PubMed  Google Scholar 

  • Seelig LL Jr, Doody P, Brainard L, Gidda JS, Goyal RK (1984) Acetylcholinesterase and choline acetyltransferase staining of neurons in the opossum esophagus. Anat Rec 209(1):125–130. doi:10.1002/ar.1092090115

    Article  PubMed  Google Scholar 

  • Singaram C, Sengupta A, Sweet MA, Sugarbaker DJ, Goyal RK (1994) Nitrinergic and peptidergic innervation of the human oesophagus. Gut 35(12):1690–1696

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Smid SD, Blackshaw LA (2000) Vagal ganglionic and nonadrenergic noncholinergic neurotransmission to the ferret lower oesophageal sphincter. Auton Neurosci 86(1–2):30–36

    Article  CAS  PubMed  Google Scholar 

  • Stach W (1981) Zur neuronalen Organisation des Plexus myentericus Auerbach im Schweinedünndarm II Typ II-neurone. Z Mikrosk Anat Forsch 95:161–192

    CAS  PubMed  Google Scholar 

  • Stick JA, Derksen FJ, McNitt DL, Chou CC (1983) Equine esophageal pressure profile. Am J Vet Res 44(2):272–275

    CAS  PubMed  Google Scholar 

  • Teixeira AF, Vives P, Krammer HJ, Kuhnel W, Wedel T (2001) Structural organization of the enteric nervous system in the cattle esophagus revealed by wholemount immunohistochemistry. Ital J Anat Embryol 106(2 Suppl 1):313–321

    CAS  PubMed  Google Scholar 

  • Timmermans JP, Scheuermann DW, Stach W, Adriaensen D, De Groodt-Lasseel MH (1992) Functional morphology of the enteric nervous system with special reference to large mammals. Eur J Morphol 30(2):113–122

    CAS  PubMed  Google Scholar 

  • van Ginneken C, Weyns A, van Meir F, Ooms L, Verhofstad A (1996) Intrinsic innervation of the stomach of the fetal pig: an immunohistochemical study of VIP-immunoreactive nerve fibres and cell bodies. Anat Histol Embryol 25(4):269–275

    Article  PubMed  Google Scholar 

  • Vanden Berghe P, Coulie B, Tack J, Mawe GM, Schemann M, Janssens J (1999) Neurochemical coding of myenteric neurons in the guinea-pig antrum. Cell Tissue Res 297(1):81–90

    Article  CAS  PubMed  Google Scholar 

  • Wedel T, Roblick U, Gleiss J, Schiedeck T, Bruch HP, Kuhnel W, Krammer HJ (1999) Organization of the enteric nervous system in the human colon demonstrated by wholemount immunohistochemistry with special reference to the submucous plexus. Ann Anat 181(4):327–337

    Article  CAS  PubMed  Google Scholar 

  • Worl J, Neuhuber WL (2005) Enteric co-innervation of motor endplates in the esophagus: state of the art ten years after. Histochem Cell Biol 123(2):117–130. doi:10.1007/s00418-005-0764-7

    Article  PubMed  Google Scholar 

  • Wu M, Van Nassauw L, Kroese AB, Adriaensen D, Timmermans JP (2003) Myenteric nitrergic neurons along the rat esophagus: evidence for regional and strain differences in age-related changes. Histochem Cell Biol 119(5):395–403. doi:10.1007/s00418-003-0526-3

    CAS  PubMed  Google Scholar 

  • Yuan S, Costa M, Brookes SJ (1998) Neuronal pathways and transmission to the lower esophageal sphincter of the guinea Pig. Gastroenterology 115(3):661–671

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Chiara Bernardini for their technical assistance. This study was supported in part by a Grant (2010bis/2011) from the Fondazione del Monte di Bologna e Ravenna.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Veterinary Medical Sciences (UNI EN ISO 9001:2008), University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell’Emilia, Bologna, Italy

    R. Chiocchetti, F. Giancola, M. Mazzoni, C. Sorteni, N. Romagnoli & M. Pietra

Authors
  1. R. Chiocchetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F. Giancola
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Mazzoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Sorteni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N. Romagnoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. Pietra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. Chiocchetti.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chiocchetti, R., Giancola, F., Mazzoni, M. et al. Excitatory and inhibitory enteric innervation of horse lower esophageal sphincter. Histochem Cell Biol 143, 625–635 (2015). https://doi.org/10.1007/s00418-014-1306-y

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-014-1306-y

Keywords

  • Choline acetyltransferase
  • Enteric nervous system
  • Horse
  • Immunohistochemistry
  • Neuronal nitric oxide synthase