Myenteric Neuronal Cell Bodies Research Articles

Localization of Substance P-Immunoreactive Nerve Structures in the Rat Colon

The tachykinins, including their major representative substance P (SP), are an essential component of its non-adrenergic, non-cholinergic transmission of the myenteric plexus of the enteric nervous system. Hence, the understanding of the role that SP plays in the conduction of a wide variety of motility and secretory reflexes lies in the evidence-based understanding of the sites of its expression. In the present study, the presence, distribution and staining intensity of the SP-positive nerve structures in the myenteric plexus of the proximal and distal colon of the adult rat have been investigated by means of immunohistochemistry at the light microscopic level. We observed an abundance of intensely stained beaded SP-immunopositive fibres, surrounding the SP-immunonegative myenteric neuronal cell bodies in a basket-like manner. Fine bundles of SP-immunostained varicose fibres were also registered in the adjacent smooth muscle layers. The statistical analysis revealed discernible prevalence of the staining intensity in the proximal colon, compared to the distal one. In summary, our findings provide immunohistochemical evidence for the occurrence of abundant SP-immunopositive nerve structures in the colonic region of the rat. Taken together with previous morphological and pharmacological research the results of this study suggest that the SP-mediated neurotransmission is of physiological and likely of pathological significance for the examined region.

Neurochemical profile of the myenteric plexus in the rat colorectal region.

The enteric nervous system, a major subdivision of the autonomic nervous system, is known for its neurochemical heterogeneity and complexity. The myenteric plexus, one of its two principal components, primarily controls peristalsis and its dysfunction may lead to a number of gastrointestinal motility disorders. The myenteric neurons have been described to use a wide variety of neurotransmitters although no evidence has been reported for the existence of adrenergic neurons in the hindgut. This study aims at elucidating the chemical coding of neurons in the myenteric plexus of the rat colon and anorectal region with particular emphasis on cholinergic and the so-called nonadrenergic, noncholinergic (NANC) transmitter systems. The immunostaining for choline acetyltransferase revealed an intense staining of the myenteric ganglia with clear delineation of their neuronal cell bodies and without local distributional differences in the colonic region. The myenteric ATPergic structures were mostly limited to fiber bundles surrounding unstained myenteric neurons and penetrating the two muscle layers. We also observed an abundance of intensely stained varicose substance P-immunopositive fibers, ensheathing the immunonegative myenteric neuronal cell bodies in a basket-like manner. Applying NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry, we were able to demonstrate numerous nitrergic somata of myenteric neurons with Dogiel Type I morphology. Apart from the observed nitrergic distributional patterns, no distinct variations were found in the staining intensity or distribution of myenteric structures in the colon and anorectal area. Our results suggest that myenteric neurons in the distal intestinal portion utilize a broad spectrum of enteric transmitters, including classical and NANC transmitters.

Паракватная модель паркинсонизма и выявление фосфорилированного α-синуклеина в энтеральной нервной системе у крыс

Parkinson’s disease (PD) is a common neurodegenerative disorder with a variety of motor and non-motor features. Non-motor symptoms, such as gastrointestinal dysfunction, usually set in 5 to 15 years earlier than motor manifestations. Cytoplasmic aggregates of phosphorylated α-synuclein are a typical marker of PD. They are observed not only in cerebral neurons but also in intramural plexuses of the intestine. Therefore, it is essential to investigate the peripheral component of the molecular pathogenesis of the disease using PD models, including those involving the use of parkinsonian neurotoxins, such as the well-known herbicide paraquat. The aim of this study was to identify a complex of early α-synuclein-related changes induced by long-term systemic administration of paraquat to rats at doses of 6 mg/kg. The open-field test revealed a decline in the motor activity of the experimental animals; the tapered beam walking test demonstrated a two-fold increase (р = 0.044) in the number of left paw slips. Besides, the intensity of staining for tyrosine hydroxylase (TH) in the substantia nigra and myenteric plexus fibers was 50% (р = 0.033) and 20% (р = 0.01) lower, respectively, in the main group than in the controls. Phosphorylated α-synuclein content was increased in the cell bodies of myenteric neurons and in TH-positive nervous fibers of the experimental animals. Changes indicating the development of peripheral α-synuclein pathology in the early stage of induced PD are similar to the changes observed in patients with PD at the onset of the disease. The proposed paraquat regimen could be very promising for PD modeling.

Homer1 (VesL-1) in the rat esophagus: focus on myenteric plexus and neuromuscular junction.

Homer1, a scaffolding protein of the postsynaptic density (PSD), enriched at excitatory synapses is known to anchor and modulate group I metabotropic glutamate receptors (mGluRs) and different channel- and receptor-proteins. Homer proteins are expressed in neurons of different brain regions, but also in non-neuronal tissues like skeletal muscle. Occurrence and location of Homer1 and mGluR5 in myenteric plexus and neuromuscular junctions (NMJ) of rat esophagus have yet not been characterized. We located Homer1 and mGluR5 immunoreactivity (-iry) in rat esophagus and focused on myenteric neurons, intraganglionic laminar endings (IGLEs) and NMJs, using double- and triple-label immunohistochemistry and confocal laser scanning microscopy. Homer1-iry was found in a subpopulation of vesicular glutamate transporter 2 (VGLUT2) positive IGLEs and cholinergic varicosities within myenteric ganglia, but neither in nitrergic nor cholinergic myenteric neuronal cell bodies. Homer1-iry was detected in 63% of esophageal and, for comparison, in 35% of sternomastoid NMJs. Besides the location in the PSD, Homer1-iry colocalized with cholinergic markers, indicating a presynaptic location in coarse VAChT/CGRP/NF200- immunoreactive (-ir) terminals of nucleus ambiguus neurons supplying striated esophageal muscle. mGluR5-iry was found in subpopulations of myenteric neuronal cell bodies, VGLUT2-ir IGLEs and cholinergic varicosities within the myenteric neuropil and NMJs of esophagus and sternomastoid muscles. Thus, Homer1 may anchor mGluR5 at presynaptic sites of cholinergic boutons at esophageal motor endplates, in a small subpopulation of VGLUT2-ir IGLEs and cholinergic varicosities within myenteric ganglia possibly modulating Ca2+-currents and neurotransmitter release.

Creatine supplementation in trained rats causes changes in myenteric neurons and intestinal wall morphometry

Creatine is widely used by athletes as an ergogenic resource. The aim of this study was to evaluate the influence of creatine supplementation on the duodenum of rats submitted to physical training. The number and myenteric neuronal cell bodies as well mucosal and muscular tunic morphometry were evaluated. Control animals received a standard chow for 8 weeks, and the treated ones received the standard chow for 4 weeks and were later fed with the same chow but added with 2% creatine. Animals were divided in groups: sedentary, sedentary supplemented with creatine, trained and trained supplemented with creatine. The training consisted in treadmill running for 8 weeks. Duodenal samples were either processed for whole mount preparations or for paraffin embedding and hematoxylin-eosin staining for histological and morphometric studies of the mucosa, the muscular tunic and myenteric neurons. It was observed that neither creatine nor physical training alone promoted alterations in muscular tunic thickness, villus height or crypts depth, however, a reduction in these parameters was observed when both were associated. The number of myenteric neurons was unchanged, but the neuronal cell body area was reduced in trained animals but not when training and creatine was associated, suggesting a neuroprotector role of this substance.

Neurochemical coding compared between varicose axons and cell bodies of myenteric neurons in the guinea-pig ileum

The discrete functional classes of enteric neurons in the mammalian gastrointestinal tract have been successfully distinguished on the basis of the unique combination of molecules and enzymes in their cell bodies (“chemical coding”). Whether the same chemical coding exists in varicose axons of different functional classes has not been systematically tested. In this study, we quantified the coexistence of markers that define classes of nerve cell bodies in the myenteric plexus of the guinea-pig ileum, in varicose axons of the same neurons. Profound differences between the combinations of immunohistochemical markers in myenteric nerve cell bodies and in their varicosities were identified. These discrepancies were particularly notable for classes of neurons that had previously been classified as cholinergic, based on immunoreactivity for choline acetyltransferase (ChAT) in their cell bodies. To detect cholinergic varicose axons of enteric neurons in this study, we used antiserum against the vesicular acetylcholine transporter (VAChT). ChAT-immunoreactivity has been reported to be consistently co-localized with 5-hydroxytryptamine (5-HT) in interneuronal cell bodies, yet only 29±5% (n=4) of 5-HT-immunoreactive varicosities contained vesicular acetylcholine transporter (VAChT). Somatostatin coexists with ChAT-immunoreactivity in a class of descending interneuron but only 21±1% (n=4) of somatostatin-immunoreactive varicosities were VAChT-immunoreactive. Comparable discrepancies were also noted for non-cholinergic markers. The results suggest that chemical coding of cell bodies does not necessarily reflect chemical coding of varicose axon terminals and that the assumption that nerve cell bodies that contain ChAT are functionally cholinergic may be questionable.

Serotonin receptor diversity in the human colon: Expression of serotonin type 3 receptor subunits 5‐HT3C, 5‐HT3D, and 5‐HT3E

Since the first description of 5-HT₃ receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown that these subunits are able to form functional heteromeric receptors when coexpressed with the 5-HT3A subunit. However, whether these subunits are actually expressed in human tissue remained to be confirmed. In the current study, we performed immunocytochemistry to locate the 5-HT3A as well as the 5-HT3C, 5-HT3D, and 5-HT3E subunits within the human colon. Western blot analysis was used to confirm subunit expression, and RT-PCR was employed to detect transcripts encoding 5-HT₃ receptor subunits in microdissected tissue samples. This investigation revealed, for the first time, that 5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons. Furthermore, 5-HT3A and 5-HT3D were found to be expressed in submucosal plexus of the human large intestine. These data provide a strong basis for future studies of the roles that specific 5-HT₃ receptor subtypes play in the function of the enteric and central nervous systems and the contribution that specific 5-HT₃ receptors make to the pathophysiology of gastrointestinal disorders such as irritable bowel syndrome and dyspepsia.

Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus

In rat and mouse esophagus, vesicular glutamate transporter 2 (VGLUT2) has been demonstrated to identify vagal intraganglionic laminar endings (IGLEs); this has recently also been shown for VGLUT1 in rat esophagus. In this study, we have investigated the distribution of VGLUT1 in the mouse esophagus and compared these results with the recently published data from the rat esophagus. Unexpectedly, we have discovered that VGLUT1 mostly fails to identify IGLEs in the mouse esophagus. This is surprising, since the distribution of VGLUT2 shows comparable results in both species. Confocal imaging has revealed substantial colocalization of VGLUT1 immunoreactivity (-ir) with cholinergic and nitrergic/peptidergic markers within the myenteric neuropil and in both cholinergic and nitrergic myenteric neuronal cell bodies. VGLUT1 and cholinergic markers have also been colocalized in fibers of the muscularis mucosae, whereas VGLUT1 and nitrergic markers have never been colocalized in fibers of the muscularis mucosae, although this does occur in fibers of the muscularis running to motor endplates. Thus, VGLUT1 is contained in the nitrergic innervation of mouse esophageal motor endplates, another difference from the rat esophagus. VGLUT1-ir is therefore present in extrinsic and intrinsic innervation of the mouse esophagus, but the significant differences from the rat indicate species variations concerning the distribution of VGLUTs in the peripheral nervous system.

Myenteric denervation differentially reduces enteroendocrine serotonin cell population in rats during postnatal development

The enteric nervous and enteroendocrine systems regulate different processes in the small intestine. Ablation of myenteric plexus with benzalkonium chloride (BAC) stimulates epithelial cell proliferation, whereas endocrine serotonin cells may inhibit the process. To evaluate the connection between the systems and the influence of myenteric plexus on serotoninergic cells in rats during postnatal development, the ileal plexus was partially removed with BAC. Rats were treated at 13 or 21 days and sacrificed after 15 days. The cell bodies of myenteric neurons were stained by beta NADH-diaphorase to detect the extension of denervation. The number of enteroendocrine cells in the ileum was estimated in crypts and villi in paraffin sections immunostained for serotonin. The number of neurons was reduced by 27.6 and 45% in rats treated on the 13th and 21st days, respectively. We tried to establish a correlation of denervation and the serotonin population according to the age of treatment. We observed a reduction of immunolabelled cells in the crypts of rats treated at 13 days, whereas this effect was seen in the villi of rats denervated at 21 days. These results suggest that the enteric nervous system might control the enteroendocrine cell population and this complex mechanism could be correlated to changes in cell proliferation.

Vesicular glutamate transporter 1 immunoreactivity in extrinsic and intrinsic innervation of the rat esophagus

Encouraged by the recent finding of vesicular glutamate transporter 2 (VGLUT2) immunoreactivity (-ir) in intraganglionic laminar endings (IGLEs) of the rat esophagus, we investigated also the distribution and co-localization patterns of VGLUT1. Confocal imaging revealed substantial co-localization of VGLUT1-ir with selective markers of IGLEs, i.e., calretinin and VGLUT2, indicating that IGLEs contain both VGLUT1 and VGLUT2 within their synaptic vesicles. Besides IGLEs, we found VGLUT1-ir in both cholinergic and nitrergic myenteric neuronal cell bodies, in fibers of the muscularis mucosae, and in esophageal motor endplates. Skeletal neuromuscular junctions, in contrast, showed no VGLUT1-ir. We also tested for probable co-localization of VGLUT1-ir with markers of extrinsic and intrinsic esophageal innervation and glia. Within the myenteric neuropil we found, besides co-localization of VGLUT1 and substance P, no further co-localization of VGLUT1-ir with any of these markers. In the muscularis mucosae some VGLUT1-ir fibers were shown to contain neuronal nitric oxide synthase (nNOS)-ir. VGLUT1-ir in esophageal motor endplates was partly co-localized with vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT)-ir, but VGLUT1-ir was also demonstrated in separately terminating fibers at motor endplates co-localized neither with ChAT/VAChT-ir nor with nNOS-ir, suggesting a hitherto unknown glutamatergic enteric co-innervation. Thus, VGLUT1-ir was found in extrinsic as well as intrinsic innervation of the rat esophagus.

Combined determination with functional and morphological studies of origin of nerve fibers expressing transient receptor potential vanilloid 1 in the myenteric plexus of the rat jejunum

The aim of this study was to determine the action of capsaicin in isolated rat intestine and the origin of nerve fibers expressing transient receptor potential vanilloid 1 (TRPV1: capsaicin receptor) in the rat jejunum by combination of functional and immunohistochemical experiments. Capsaicin (1 μM) produced a prolonged relaxation response (52.8±15.3% of the relaxation response to papaverine, mean±S.D., n=27) of the isolated jejunum in the presence of atropine and guanethidine. Pretreatment with the TRPV1 antagonist, capsazepine (10 μM) and ruthenium red (3 μM) significantly reduced the relaxation response to capsaicin by 78% ( P<0.01) and 38% ( P<0.05), respectively. Tetrodotoxin and calcitonin gene-related peptide (CGRP)-desensitization significantly reduced the response to capsaicin by 72% ( P<0.01) and 42% ( P<0.01), respectively. Therefore, we investigated the distribution of TRPV1-immunoreactivity (IR) in the myenteric plexus of the rat jejunum. Using antisera raised against either the N-terminal or C-terminal domains of rat TRPV1, TRPV1-IR was present in the nerve fibers, but not in the cell bodies of myenteric neurons. These TRPV1-immunoreactive nerve fibers were running in myenteric ganglia and their interconnecting strands. Most TRPV1-immunoreactive nerve fibers showed CGRP-IR, whereas few VR1-immunoreactive nerve fibers showed substance P-IR. After chronic denervation of the extrinsic nerve supply to the jejunum, both the relaxation response to capsaicin and TRPV1-immunoreactive nerve fibers completely disappeared. These findings indicate that these TRPV1-immunoreactive nerve fibers in the rat jejunum derive from extrinsic neurons and that activation of TRPV1 produces the relaxation response in the rat jejunum, at least in part, through the release of CGRP from nerve fibers expressing TRPV1.

Streptozotocin-induced diabetes duration is important to determine changes in the number and basophily of myenteric neurons.

The aim of present study was to evaluate the number and basophily of cell bodies of myenteric neurons in the ileum of rats with diabetes mellitus induced by streptozotocin. Four groups of rats were used: diabetes was induced in two (D) whereas the other two worked as controls (N). Animals were sacrificed six (6N, 6D) or nineteen (19N, 19D) weeks after diabetes induction. A segment of the terminal portion of the ileum of each rat was obtained and stained with Giemsa's solution, for whole-mount preparation studies. Forty fields were analyzed in each animal, and the number and basophily intensity of cell bodies were recorded. After counting, the following mean numbers of neurons/mm2 were obtained: 6N=593.1 +/- 95.75, 6D=639.1 +/- 130.8, 19N=580.1 +/- 175.6 and 19D=402.0 +/- 144.8. The analysis of basophily shown that highest frequency of neurons with weak/intermediary basophily was verified in 6D group (55.3%), whereas the groups 6N, 19N e 19D presented 38%, 36% e 40% respectively. The statistical analysis showed that a long period is necessary to decrease the number of neurons/mm2 in the rat ileum after diabetes induction, and that there was a reduction in basophily intensity in diabetic rats after 6 weeks of treatment, and such cells do not recover after a longer period (19 weeks).

Immunohistochemical demonstration of GABAergic neurons in the enteric nervous system

Application of a highly specific antiserum against GABA to whole-mount preparations of the guinea pig and rat myenteric plexus resulted in discrete and unambiguous immunolabeling of a subpopulation of myenteric neuronal cell bodies and fibers. The anti-GABA antiserum, which was raised against GABA conjugated by glutaraldehyde to BSA, was applied to glutaraldehyde-fixed whole mounts and subsequently visualized using the peroxidase-antiperoxidase method. In the guinea pig ileum and colon, immunoreactive varicose nerve fibers and scattered nerve cell bodies were found within the myenteric plexus. Immunostained fibers were also seen in the tertiary plexus and in the circular muscle, running parallel to the muscle bundles. GABA immunoreactivity in these intramuscular nerves was most pronounced in the colon. In the rat, immunoreactive fibers were prominent throughout the myenteric plexus. They formed dense networks within the myenteric ganglia, which also contained immunopositive nerve cell bodies, and ran between them in the interconnecting nerves. Some immunoreactive nerve fibers were seen in the circular muscle. Control experiments using non-immune sera or adsorbed anti-GABA antiserum showed no staining. These results add a definitive support to our previous suggestion that GABA serves as an autonomic neurotransmitter in vertebrates. In addition to the present immunohistochemical evidence, this hypothesis is supported by biochemical, autoradiographic, transmitter release, electrophysiological, and pharmacological studies on the enteric nervous system of several species. It is now important to determine the functional role of GABAergic neurons within the complex neuronal circuitry that controls gut functions.

α-Melanotropin, β-endorphin and adrenocorticotropin-like immunoreactivities are colocalized within duodenal myenteric plexus perikarya

The opioid peptide β-endorphin co-exists with α-melanotropin and adrenocorticotropin within myenteric neuronal cell bodies of the rat duodenum. Adjacent serial sections through the myenteric and submucous plexus have been stained alternately with antisera directed against α-melanotropin, β-endorphin and adrenocorticotropin. This resulted in nearly superimposable immunofluorescences for the 3 peptides within neuronal cell bodies of the myenteric plexus. An α-melanotrophin staining was always linked by an immunofluorescence for β-endorphin, adrenocorticotropin and vice versa. These staining patterns were not seen in the submucous plexus. Some tangentially cut nerve fibers running through the longitudinal muscle layer revealed a coexistence of adrenocorticotropin and α-melanotropin. The colocalization of β-endorphin, α-melanotropin and adrenocorticotropin within the same perikarya may reflect a physiological role for the 3 peptides in the nervous system of the rat duodenum.