The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum

doi:10.1016/j.neuroscience.2009.12.034

Neuroscience

Volume 166, Issue 2, 17 March 2010, Pages 564-579

https://doi.org/10.1016/j.neuroscience.2009.12.034 Get rights and content

Abstract

In vitro anterograde tracing of axons in mesenteric nerve trunks using biotinamide in combination with immunohistochemical labelling was used to characterize the extrinsic nerve projections in the myenteric plexus of the mouse jejunum. Anterogradely-labelled spinal sensory fibres innervating the enteric nervous system were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP), while sympathetic noradrenergic fibres were detected with tyrosine hydroxylase (TH), using confocal microscopy. The presence of these markers has been previously described in the spinal sensory and sympathetic fibres. Labelled extrinsic nerve fibres in the myenteric plexus were identified apposing enteric neurons that were immunoreactive for either calretinin (CalR), calbindin (CalB) or nitric oxide synthase (NOS). Of the total anterogradely labelled axons in the myenteric plexus, 20% were CGRP-immunoreactive. Labelled CGRP-immunoreactive varicosities were closely apposed to CalR-immunoreactive myenteric cells, many of which were Dogiel type I (40%; interneurons) or type II (20%; intrinsic sensory) neurons. Labelled CGRP-immunoreactive varicosities were also observed in close appositions to CalB-immunoreactive myenteric cell bodies, of which a small subset had type II morphology (18%; intrinsic sensory neurons). A further 43% of all biotinamide-filled fibres were immunoreactive for TH and these fibres were apposed to CalR-immunoreactive cell bodies (small-sized; excitatory motor neurons) and NOS-immunoreactive cell bodies (either type I or small neurons; inhibitory motor neurons and interneurons) in the myenteric plexus. The results provide a neurochemical and neuroanatomical basis for connections between dorsal root afferent neurons and myenteric neurons and suggest an anatomical substrate for the well-known modulation of enteric circuits from sympathetic nerves. No anterogradely-labelled fibres were stained for NOS-immunoreactivity, despite more than 60% of dorsal root ganglion (DRG) neurons retrogradely labelled from the jejunum showing NOS-immunoreactivity. This was due to a substantial, time-dependent, and apparently selective, loss of NOS from extrinsic axons under in vitro conditions. Lastly, a small population of non-immunoreactive biotinamide-filled fibres (<1%) gave rise to dense terminal structures around individual myenteric cell bodies lacking CalR, CalB or NOS. These specialized endings may represent vagal fibres or a subset of spinal sensory neurons that do not contain CGRP.

Section snippets

Experimental procedures

Eleven adult C57Bl/6 mice (eight male; three female) weighing between 20 and 30 g were used in this study. All experimental procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee (AEEC #06133) in accordance with the ethical guidelines of the Australian code of practice for the care and use of animals for scientific purposes. The animals were maintained under standard conditions on a 12 h light/dark cycle with food and water available ad libitum.

General observations

Application of biotinamide to the mesenteric nerves of isolated sections of jejunum in vitro produced intense filling of extrinsic axons and their terminal ramifications and structures within the myenteric ganglia of the jejunum. Fibres were observed running in the mesentery to enter the mesenteric edge of the preparation and through the ganglia and internodal strands of the myenteric plexus. The complete filling of anterogradely-labelled nerve fibres allowed individual fibres to be readily

Discussion

This is the first study to describe the distribution and projections of identified dorsal root afferent axons within the myenteric plexus of the jejunum of any mammal. The processes of dorsal root afferents typically ran within only 1–2 mm of the mesenteric border in the myenteric plexus, which contrasts with the processes of sympathetic neurons which can project up to half way around the circumference of the jejunum within the myenteric plexus (Fig. 3). Neither class of extrinsic axon branches

Conclusion

The data presented here provide an anatomical and neurochemical basis for a connection between dorsal root afferent axons and myenteric neurons in the mouse jejunum. While the sensory modalities that excite these extrinsic primary afferents have yet to be determined, it is likely that these contacts can be a route by which the dorsal root axons can modulate intestinal circuits without the involvement of the CNS (Fig. 9). The results also suggest sites at which sympathetic axons may exert their

Acknowledgments

This work was supported in part by a grant (400053) from the National Health and Medical Research Council of Australia.

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As discussed below, after a consideration of the normal adult profile of jejunal catecholaminergic fibers, the patterns of age-related remodeling and the macrophage distributions associated with them suggest some mechanisms that could be involved in the axonopathies of gut aging. The basic patterns of sympathetic innervation observed in the healthy 3–8-month-old adult rat jejunums (and, notably, even in the 16–24-month-old aged rats, in unaffected jejunal sites where no axonopathies were observed) were comparable to those described in a report on the extrinsic innervation of the adult mouse jejunum (Tan et al., 2010). Specifically, TH-IR axons extensively innervate the myenteric plexus and the smooth muscle layers of the muscularis.
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In the latter, however, most of the several retrogradely traced neurons from the urinary bladder lacked TH and NET-1, possibly representing cholinergic input, as previously shown in studies on rat (Keast et al., 1995) and guinea pig (Elfvin et al., 1997). However, in a recent study in mouse, Tan et al. (2010) suggested that some TH-IR fibers apparently contacting jejunal myenteric plexus neurons, and with a confirmed extrinsic origin, could have a sensory origin. Our present results in mouse support the hypothesis of TH-expressing visceral sensory neurons as being the origin of at least a fraction of nerve fibers innervating the colorectum or the urinary bladder.
Previous studies in rat and mouse documented that a subpopulation of dorsal root ganglion (DRG) neurons innervating non-visceral tissues express tyrosine hydroxylase (TH). Here we studied whether or not mouse DRG neurons retrogradely traced with Fast Blue (FB) from colorectum or urinary bladder also express immunohistochemically detectable TH. The lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) were included in the analysis. Previously characterized antibodies against TH, norepinephrine transporter type 1 (NET-1) and calcitonin gene-related peptide (CGRP) were used. On average, ∼14% of colorectal and ∼17% of urinary bladder DRG neurons expressed TH and spanned virtually all neuronal sizes, although more often in the medium-sized to small ranges. Also, they were more abundant in lumbosacral than thoracolumbar DRGs, and often coexpressed CGRP. We also detected several TH-immunoreactive (IR) colorectal and urinary bladder neurons in the LSC and the MPG, more frequently in the former. No NET-1-IR neurons were detected in DRGs, whereas the majority of FB-labeled, TH-IR neurons in the LSC and MPG coexpressed this marker (as did most other TH-IR neurons not labeled from the target organs). TH-IR nerve fibers were detected in all layers of the colorectum and the urinary bladder, with some also reaching the basal mucosal cells. Most TH-IR fibers in these organs lacked CGRP. Taken together, we show: (1) that a previously undescribed population of colorectal and urinary bladder DRG neurons expresses TH, often CGRP but not NET-1, suggesting the absence of a noradrenergic phenotype; and (2) that TH-IR axons/terminals in the colon or urinary bladder, naturally expected to derive from autonomic sources, could also originate from sensory neurons.

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NeuroanatomyResearch PaperThe neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum

Abstract

Section snippets

Experimental procedures

General observations

Discussion

Conclusion

Acknowledgments

Neuroscience

Prog Neurobiol

Exp Neurol

Prog Neurobiol

Auton Neurosci

Prog Neurobiol

J Chem Neuroanat

J Chem Neuroanat

Neuroscience

Neuroscience

J Auton Nerv Syst

Gastroenterology

J Chem Neuroanat

Neuroscience

Neuroscience

Neuroscience

J Auton Nerv Syst

Neuroscience

Neuroscience

Gastroenterology

Brain Res

Brain Res

Eur J Pharmacol

Neuroscience

Auton Neurosci

J Neurosci Methods

J Chem Neuroanat

Neuroanatomy of extrinsic afferents supplying the gastrointestinal tract

Neurogastroenterol Motil

Topography of efferent vagal innervation of the rat gastrointestinal tract

Am J Physiol

Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract

Anat Embryol

Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor

J Comp Neurol

Analysis of the responses of myenteric neurons in the small intestine to chemical stimulation of the mucosa

Am J Physiol

Influeunce of the pattern of jejunal distension on mesenteric afferent sensitivity in the anaesthetized rat

Neurogastroenterol Motil

Sensory innervation of the viscera—peripheral basis of visceral pain

Physiol Rev

Neuroanatomy
Research Paper
The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum