Skip to main content
SpringerLink
Log in

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BSA:

Bovine serum albumin

CGRP:

calcitonin generelated peptide

DiA:

carbocyanine dye A

DiI:

carbocyanine dye I

dmnX:

dorsal motor nucleus of vagus

DMSO:

dimethylsulfoxide

ENK:

enkephalin

FITC:

fluorescin isothiocyanate

NADPH:

diaphorase nicotinamide adenine diphosphate

NPY:

neuropeptide Y

NTS:

nucleus tractus solitarii

PBS:

phosphate-buffered saline

VIP:

vasoactive intestinal peptide

WGA-HRP:

wheat-germ agglutinine-horseradish peroxidase

References

  • Allescher HD, Daniel EE (1988) Extrinsic and intrinsic neural control of pylorus sphincter pressure in the dog. J Physiol (Lond) 401:17–38

    Article  CAS  Google Scholar 

  • Alumets J, Fahrenkrug J, Håkanson R, Schaffalitzky de Muckadell O, Sundler F, Uddman R (1979) A rich VIP nerve supply is characteristic of sphincters. Nature 280:155–156

    Article  CAS  PubMed  Google Scholar 

  • Anuras S, Cooke AR, Christensen J (1974) An inhibitory innervation at the gastroduodenal junction. J Clin Invest 54:529–535

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Behar J, Biancani P, Zabinski MP (1979) Characterization of feline gastroduodenal junction by neural and hormonal stimulation. Am J Physiol 236: E45-E51

    CAS  PubMed  Google Scholar 

  • Berezin I, Huizinga JD, Daniel EE (1988) Interstitial cells of Cajal in the canine colon: A special communication network at the inner border of the circular muscle. J Comp Neurol 273:42–51

    Article  CAS  PubMed  Google Scholar 

  • Berthoud HR, Powley TL (1991) Morphology and distribution of efferent vagal innervation of rat pancreas as revealed with anterograde transport of DiI. Brain Res 553:336–341

    Article  CAS  PubMed  Google Scholar 

  • Berthoud HR, Powley TL (1992) Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor. J Comp Neurol 319:261–276

    Article  CAS  PubMed  Google Scholar 

  • Berthoud HR, Kressel M, Neuhuber WL (1992a) An anterograde tracing study of the vagal innervation of rat liver, portal vein and biliary system. Anat Embryol 186:341–442

    Article  Google Scholar 

  • Berthoud HR, Kressel M, Neuhuber WL (1992b) Anatomical localization and structure of putative vagal nutrient and osmoreceptors in liver and duodenum. Proceedings Society for the study of Ingestive Behavior, Princeton, NJ, p 79

  • Berthoud HR, Jedrzejewska A, Powley TL (1990) Simultaneous innervation of vagal innervation of gut and afferent projections from the visceral forebrain with DiI injected into the dorsal vagal complex in the rat. J Comp Neurol 301:65–79

    Article  CAS  PubMed  Google Scholar 

  • Berthoud HR, Carlson NR, Powley TL (1991) Topography of efferent vagal innervation of rat gastrointestinal tract. Am J Physiol 260:R200-R207

    CAS  PubMed  Google Scholar 

  • Bult H, Boeckxstaens GE, Pelckmans PA, Jordaens FH, Van Maercke YM, Herman AG (1990) Nitric oxide as an inhibitory non-adrenergic noncholinergic neurotransmitter. Nature 345:346–347

    Article  CAS  PubMed  Google Scholar 

  • Cai W-Q, Gabella G (1984) Structure and innervation of the musculature at the gastroduodenal junction of the guinea pig. J Anat 139:93–104

    PubMed  PubMed Central  Google Scholar 

  • Cooke AR (1975) Control of gastric emptying and motility. Gastroenterology 68:804–816

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Daniel EE, Posey-Daniel V (1984) Neuromuscular structures in opossum esophagus: role of interstitial cells of Cajal. Am J Physiol 246: G305-G315

    CAS  PubMed  Google Scholar 

  • Daniel EE, Berezin I, Allescher HD, Manaka H, Posey-Daniel V (1989) Morphology of the canine pyloric sphincter in relation to function. Can J Physiol Pharmacol 67:1560–1573

    Article  CAS  PubMed  Google Scholar 

  • Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 88:7797–7801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Desai KM, Zembowicz A, Sessa WC, Vane JR (1991) Nitroxergic nerves mediate vagally induced relaxation in the isolated stomach of the guinea pig. Proc Natl Acad Sci 88:11490–11491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dockray GJ (1991) Mediation and modulation of gastric afferent functions by regulatory peptides. In: Y Tache, D Wingate (eds) Brain-Gut interactions. CRC Press, Boca Raton, pp 123–132

    Google Scholar 

  • Edin R, Lundberg J, Terenius L, Dahlström A, Hökfelt T, Kewenter J, Ahlman H (1980) Evidence for vagal enkephalinergic neural control of the feline pylorus and stomach. Gastroenterology 78:492–497

    Article  CAS  PubMed  Google Scholar 

  • Edin R (1980) The vagal control of the pyloric motor function. A physiological and immunohistochemical study in cat and man. Acta Physiol Scand 485:1–30

    CAS  Google Scholar 

  • Edin R, Lundberg JM, Ahlman H, Dahlström A, Fahrenkrug J, Hökfelt T, Kewenter J (1979) On the VIP-ergic innervation of the feline pylorus. Acta Physiol Scand 185–187

  • Ehrlein HJ (1988) Motility of the pyloric sphincter studied by the inductograph method in conscious dogs. Am J Physiol 254:G650-G657

    CAS  PubMed  Google Scholar 

  • Elfvin LG, Lindh B (1982) A study of the extrinsic innervation of the guinea pig pylorus with the horseradish peroxidase tracing technique. J Comp Neurol 208:317–324

    Article  CAS  PubMed  Google Scholar 

  • Fisher R, Cohen S (1973) Physiological characteristics of the human pyloric sphineter. Gastroenterology 64:67–75

    Article  CAS  PubMed  Google Scholar 

  • Furness JB, Costa M, Franco R, Llewellyn-Smith IJ (1980) Neuronal peptides in the intestine: distribution and possible functions. In: E Costa, M Trabucchi (eds) Neural peptides and neuronal communication. Raven Press, New York, pp 601–617

    Google Scholar 

  • Gonda T, Daniel EE, McDonald TJ, Fox JET, Brooks BD, Oki M (1989) Distribution and function of enteric GAL-IR nerves in dogs: comparison with VIP. Am J Physiol 256:G884-G896

    CAS  PubMed  Google Scholar 

  • Grundy D (1988) Speculations on the structure/function relationship for vagal and splanchnic afferent endings supplying the gastrointestinal tract. J Auton Nerv Syst 22:175–180

    Article  CAS  PubMed  Google Scholar 

  • Holle GE, Hahn D, Forth W (1992) Innervation of pylorus in control of motility and gastric emptying. Am J Physiol 263:G161-G168

    CAS  PubMed  Google Scholar 

  • Holzer P (1988) Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24:739–768

    Article  CAS  PubMed  Google Scholar 

  • Hope BT, Michael GJ, Knigge KM, Vincent SR (1991) Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 88:2811–2814

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Isenberg JI, Csendes A (1972) Effect of octapeptide of cholecystokinin on canine pyloric pressure. Am J Physiol 222:428–431

    Article  CAS  PubMed  Google Scholar 

  • Kirchgessner AL, Gershon MD (1989) Identification of vagal efferent fibers and putative target neurons in the enteric nervous system of the rat. J Comp Neurol 285:38–53

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi S (1990) The structure of the autonomic end apparatus in the guinea-pig small intestine and the problem of the interstitial cells of Cajal. Acta Medica et Biologica 38:103–127

    Google Scholar 

  • Lawrentjew BI (1929) Experimentell-morphologische Studien über den feineren Bau des autonomen Nervensystems. II. über den Aufbau der Ganglien der Speiseröhre nebst einigen Bemerkungen über das Vorkommen und die Verteilung zweier Arten von Nervenzellen in dem autonomen Nervensystem. Z Mikrosk Anat Forsch 18:233–262

    Google Scholar 

  • Lerman SH, Jacobowitz DM, Mason GR, Garber HI, Ormsbee HS (1981) Gastric and pyloric motor response to sympathetic nerve stimulation after chemical sympathectomy. J Auton Nerv Syst 4:207–215

    Article  CAS  PubMed  Google Scholar 

  • Lindh B, Dalsgaard C-J, Elfvin L-G, Hökfelt T, Cuello AC (1983) Evidence of substance P immunoreactive neurons in dorsal root ganglia and vagal ganglia projecting to the guinea pig pylorus. Brain Res 269:365–369

    Article  CAS  PubMed  Google Scholar 

  • McHugh PR, Moran TH (1985) The stomach: a conception of its dynamic role in satiely. Prog Psychobiol Physiolo Psychol 2:197–232

    Google Scholar 

  • Mesulam MM (1978) Tetramethylbenzidine for horseradish peroxidase neurochemistry. J Histochem Cytochem 26:106–117

    Article  CAS  PubMed  Google Scholar 

  • Morgan KG, Schmalz PF, Szurszewski JH (1978) The inhibitory effects of vasoactive intestinal polypeptide on the mechanical and electrical activity of canine antral smooth muscle. J Physiol (Lond) 282:437–450

    Article  CAS  Google Scholar 

  • Neuhuber WL (1987) Sensory vagal innervation of the rat esophagus and cardia: a light and electron microscopic anterograde tracing study. J Auton Nerv Syst 20:243–255

    Article  CAS  PubMed  Google Scholar 

  • Neuhuber WL, Clerc N (1990) Afferent innervation of the esophagus in cat and rat. In: W Zenker, WL Neuhuber (eds) The primary afferent neuron; a survey of recent morpho-functional aspects. Plenum Press, New York, pp 93–107

    Chapter  Google Scholar 

  • Nosaka S, Kamaike T, Yasunaga K (1978) Central vagal organization in rats: an electrophysiological study. Exp Neurol 60:405–419

    Article  CAS  PubMed  Google Scholar 

  • Powley TL, Berthoud HR (1991) A fluorescent labeling strategy for staining the enteric nervous system. J Neurosci Methods 36:9–15

    Article  CAS  PubMed  Google Scholar 

  • Reynolds JC, Ouyang A, Cohen S (1984) Evidence for an opiate-mediated pyloric sphincter reflex. Am J Physiol 246:G130-G136

    CAS  PubMed  Google Scholar 

  • Rumessen JJ, Thuneberg L (1982) Plexus muscularis profundus and associated interstitial cells. I. Light microscopical studies of mouse small intestine. Anat Rec 203:115–127

    Article  CAS  PubMed  Google Scholar 

  • Scherer-Singler U, Vincent SR, Kimura H, McGeer EG (1983) Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J Neurosci Methods 9:229–234

    Article  CAS  PubMed  Google Scholar 

  • Smith GT, Moran TH, Coyle JT, Kuhar MJ, O'Donahue TL, McHugh PR (1984) Anatomical localization of cholecystokinin receptors to the pyloric sphincter. Am J Physiol 246:R127-R130

    CAS  PubMed  Google Scholar 

  • Stach W (1972) Der Plexus entericus extremus des Dickdarms und seine Beziehungen zu den interstitiellen Zellen (Cajal). Z Mikrosk Anat Forsch 85:245–272

    CAS  PubMed  Google Scholar 

  • Swett JE, Bourassa CM (1981) Electrical stimulation of peripheral nerve. In: MM Patterson, RP Kesner (eds) Electrical stimulation research techniques. Academic Press, New York, pp 244–298

    Google Scholar 

  • Tack JF, Janssens W, Vantrappen G (1992) Regional neurophysiology of the enteric nervous system (abstract). Dig Dis Sci 37:964

    Google Scholar 

  • Telford GL, Mir SS, Mason GR, Ormsbee HS (1979) Neural control of the canine pylorus. Am J Surg 137:92–98

    Article  CAS  PubMed  Google Scholar 

  • Tottrup A, Svane D, Forman A (1991) Nitric oxide mediating NANC inhibition in opposum lower esophageal sphincter. Am J Physiol 260:G385-G389

    CAS  PubMed  Google Scholar 

  • Tougas G, Allescher HD, Dent J, Daniel EE (1991) Sensory nerves of the intestines: role in control of pyloric region of dogs. In: Costa M et al (eds) Sensory nerves and neuropeptides in gastroenterology, Plenum Press, New York, pp 199–211

    Chapter  Google Scholar 

  • Van der Winden J-M, Mailleux P, Schiffmann SN, Vanderhaeghen J-J, DeLaet M-H (1992) Nitric oxide synthase activity in infantile hypertrophic pyloric stenosis. N Engl J Med 327:511–515

    Article  Google Scholar 

  • Young HM, Furness JB, Shuttleworth CWR, Bredt DS, Synder SH (1992) Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea pig intestine. Histochemistry 97:375–378

    Article  CAS  PubMed  Google Scholar 

  • Zhou D-S, Komuro T (1992) Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine, with special reference to the interstitial cells of Cajal. Cell Tissue Res 268:205–216

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Anatomy Institute, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland

    Michael Kressel

  2. Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, 70808, Baton Rouge, LA, USA

    Hans-Rudolf Berthoud

  3. Anatomy Institute, University of Erlangen, Krankenhausstrasse 9, D-91054, Erlangen, Germany

    Winfried L. Neuhuber

Authors
  1. Michael Kressel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hans-Rudolf Berthoud
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Winfried L. Neuhuber
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kressel, M., Berthoud, HR. & Neuhuber, W.L. Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy. Cell Tissue Res 275, 109–123 (1994). https://doi.org/10.1007/BF00305379

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00305379

Key words

Search

Navigation