Abstract
Intraganglionic laminar endings (IGLEs) represent the major vagal afferent structures throughout the gastrointestinal tract. Previous ultrastructural investigations have revealed synaptic contacts of IGLEs on myenteric neurons. Thus, in addtion to functioning probably as mechanosensors, IGLEs may also synaptically influence myenteric neurons. In search of clues for potential transmitters in IGLEs, we investigated, by combined neuronal tracing and immunocytochemistry in the esophagus, the correlation between IGLEs and vesicular glutamate transporter 2 (VGLUT2), which is considered a reliable marker for glutamatergic neurons. In rat esophagus, IGLEs were immunostained with calretinin. In the mouse, anterograde wheat germ agglutinin/horseradish peroxidase (WGA-HRP) tracing from nodose ganglion was used in order to label esophageal IGLEs. Confocal laser scanning microscopy demonstrated that VGLUT2 immunoreactivity was highly colocalized with synaptophysin and that both calretinin and tyramide amplified WGA-HRP in rat and mouse esophagus, respectively. No colocalization was found with calcitonin gene-related peptide, a marker for spinal primary afferents. Thus, VGLUT2 is found in vagal afferent endings in the esophagus, suggesting that glutamate is contained in, and probably released from, synaptic vesicles previously described in IGLEs. Functional evidence pending, this finding is in favor of a local effector function of IGLEs onto myenteric neurons.
Similar content being viewed by others
References
Bellocchio EE, Reimer RJ, Fremeau RT Jr, Edwards RH (2000) Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289:957–960
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
Berthoud HR, Patterson LM, Neumann F, Neuhuber WL (1997) Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract. Anat Embryol (Berl) 195:183–191
Dütsch M, Eichhorn U, Wörl J, Wank M, Berthoud HR, Neuhuber WL (1998) Vagal and spinal afferent innervation of the rat esophagus: a combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins. J Comp Neurol 398:289–307
Foley CM, Moffitt JA, Hay M, Hasser EM (1998) Glutamate in the nucleus of the solitary tract activates both ionotropic and metabotropic glutamate receptors. Am J Physiol 275:R1858–R1866
Fremeau RT Jr, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, Bellocchio EE, Fortin D, Storm-Mathisen J, Edwards RH (2001) The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31:247–260
Herzog E, Bellenchi GC, Gras C, Bernard V, Ravassard P, Bedet C, Gasnier B, Giros B, El Mestikawy S (2001) The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons. J Neurosci 21:RC181
Hoang CJ, Hay M (2001) Expression of metabotropic glutamate receptors in nodose ganglia and the nucleus of the solitary tract. Am J Physiol Heart Circ Physiol 281:H457–H462
Kaneko T, Fujiyama F (2002) Complementary distribution of vesicular glutamate transporters in the central nervous system. Neurosci Res 42:243–250
Kaneko T, Fujiyama F, Hioki H (2002) Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain. J Comp Neurol 444:39–62
Keast JR, Stephensen TM (2000) Glutamate and aspartate immunoreactivity in dorsal root ganglion cells supplying visceral and somatic targets and evidence for peripheral axonal transport. J Comp Neurol 424:577–587
Kirchgessner AL, Liu MT, Alcantara F (1997) Excitotoxicity in the enteric nervous system. J Neurosci 17:8804–8816
Kressel M (1998) Tyramide amplification allows anterograde tracing by horseradish peroxidase-conjugated lectins in conjunction with simultaneous immunohistochemistry. J Histochem Cytochem 46:527–533
Kressel M, Radespiel-Tröger M (1999) Anterograde tracing and immunohistochemical characterization of potentially mechanosensitive vagal afferents in the esophagus. J Comp Neurol 412:161–172
Liu MT, Rothstein JD, Gershon MD, Kirchgessner AL (1997) Glutamatergic enteric neurons. J Neurosci 17:4764–4784
Meeley MP, Underwood MD, Talman WT, Reis DJ (1989) Content and in vitro release of endogenous amino acids in the area of the nucleus of the solitary tract of the rat. J Neurochem 53:1807–1817
Mesulam MM (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Histochem Cytochem 26:106–117
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
Neuhuber WL, Clerc N (1990) Afferent innervation of the esophagus in cat and rat. In: Zenker W, Neuhuber WL (eds) The primary afferent neuron. Plenum, New York, pp 93–107
Neuhuber WL, Wörl J, Berthoud HR, Conte B (1994) NADPH-diaphorase-positive nerve fibers associated with motor endplates in the rat esophagus: new evidence for co-innervation of striated muscle by enteric neurons. Cell Tissue Res 276:23–30
Nonidez JF (1946) Afferent nerves in the intermuscular plexus of the dog's oesophagus. J Comp Neurol 85:177–189
Perrone MH (1981) Biochemical evidence that L-glutamate is a neurotransmitter of primary vagal afferent nerve fibers. Brain Res 230:283–293
Phillips RJ, Powley TL (2000) Tension and stretch receptors in gastrointestinal smooth muscle: re-evaluating vagal mechanoreceptor electrophysiology. Brain Res Brain Res Rev 34:1–26
Reimer RJ, Fremeau RT Jr, Bellocchio EE, Edwards RH (2001) The essence of excitation. Curr Opin Cell Biol 13:417–421
Rodrigo J, Hernandez J, Vidal MA, Pedrosa JA (1975) Vegetative innervation of the esophagus. II. Intraganglionic laminar endings. Acta Anat (Basel) 92:79–100
Rodrigo J, De Felipe J, Robles-Chillida EM, Perez Anton JA, Mayo I, Gomez A (1982) Sensory vagal nature and anatomical access paths to esophagus laminar nerve endings in myenteric ganglia. Determination by surgical degeneration methods. Acta Anat (Basel) 112:47–57
Saha S, Batten TF, Mcwilliam PN (1995) Glutamate-immunoreactivity in identified vagal afferent terminals of the cat: a study combining horseradish peroxidase tracing and postembedding electron microscopic immunogold staining. Exp Physiol 80:193–202
Sang Q, Young HM (1997) Development of nicotinic receptor clusters and innervation accompanying the change in muscle phenotype in the mouse esophagus. J Comp Neurol 386:119–136
Schaffar N, Rao H, Kessler J-P, Jean A (1997) Immunohistochemical detection of glutamate in rat vagal sensory neurons. Brain Res 778:302–308
Talman WT, Perrone MH, Reis DJ (1980) Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers Science 209:813–815
Tong Q, Ma J, Kirchgessner AL (2001) Vesicular glutamate transporter 2 in the brain-gut axis. Neuroreport 12:3929–3934
Varoqui H, Schäfer MK, Zhu H, Weihe E, Erickson JD (2002) Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses. J Neurosci 22:142–155
Wang FB, Powley TL (2000) Topographic inventories of vagal afferents in gastrointestinal muscle. J Comp Neurol 421:302–324
Wiley JW, Lu YX, Owyang C (1991) Evidence for a glutamatergic neural pathway in the myenteric plexus. Am J Physiol 261:G693–G700
Wörl J, Dütsch F, Neuhuber WL (2002) Development of neuromuscular junctions in the mouse esophagus: focus on establishment and reduction of enteric co-innervation. Anat Embryol (Berl) 205:141–152
Yuan S, Brookes SJ (1999) Neuronal control of the gastric sling muscle of the guinea pig. J Comp Neurol 412:669–680
Zagorodnyuk VP, Brookes SJ (2000) Transduction sites of vagal mechanoreceptors in the guinea pig esophagus. J Neurosci 20:6249–6255
Zagorodnyuk VP, Chen BN, Brookes SJ (2001) Intraganglionic laminar endings are mechano-transduction sites of vagal tension receptors in the guinea-pig stomach. J Physiol (Lond) 534:255–268
Zheng H, Lauve A, Patterson LM, Berthoud HR (1997) Limited excitatory local effector function of gastric vagal afferent intraganglionic terminals in rats. Am J Physiol 273:G661–G669
Zhuo H, Ichikawa H, Helke CJ (1997) Neurochemistry of the nodose ganglion. Prog Neurobiol 52:79–107
Acknowledgements
The skilful technical assistance of Karin Löschner, Hedwig Symowski, Andrea Hilpert, and Inge Zimmermann is gratefully acknowledged. We especially thank Drs. A. Brehmer, M. Kressel, and F. Schrödl for helpful and stimulating discussion, Dr. W. Zimmermann for help with the Zeiss confocal microscope, and Dr. J. Wörl for providing the mice.
Author information
Authors and Affiliations
Corresponding author
Additional information
This study was supported by DFG/SFB 353, B15
Rights and permissions
About this article
Cite this article
Raab, M., Neuhuber, W.L. Vesicular glutamate transporter 2 immunoreactivity in putative vagal mechanosensor terminals of mouse and rat esophagus: indication of a local effector function?. Cell Tissue Res 312, 141–148 (2003). https://doi.org/10.1007/s00441-003-0721-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-003-0721-5