Control of peripheral glial cell proliferation: enteric neurons exert an inhibitory influence on Schwann cell and enteric glial cell DNA synthesis in culture

Neuronal membranes from rat dorsal root ganglia provide a mitogenic signal to cultured Schwann cells and it has been suggested this is an important factor in regulating Schwann cell numbers during development. In this study, the influence of enteric neurons on the DNA synthesis of both Schwann cells and enteric glia has been investigated as well as the effect of axonal membrane fractions (axolemma) on enteric glia. The proliferation rate of rat Schwann cells and enteric glia was assessed in culture using [3H]thymidine uptake and autoradiography in combination with immunolabelling to identify cell types. When purified rat Schwann cells were co-cultured with guinea pig enteric neurons, their DNA synthesis rate was reduced compared with control cultures of pure Schwann cells or Schwann cells not close to neurites or neuronal cell bodies. Nevertheless, in accordance with previous findings that sensory neurons stimulate Schwann cell division, these Schwann cells increased their DNA synthesis rate when in contact with neurites from purified guinea pig or adult rat dorsal root ganglion neurons and on exposure to bovine axolemmal fractions. The enteric neurons also suppressed the DNA synthesis of enteric glia in co-cultures of purified enteric neurons and enteric glia, while bovine axolemma stimulated their DNA synthesis. These results indicate that a mitotic inhibitory signal is associated with enteric neurons and can exert its effect on both Schwann cells and enteric glia, and that enteric glia, like Schwann cells, are stimulated to divide by axolemmal fractions. It thus seems possible that during development glial cell numbers in the peripheral nervous system may be controlled by both positive and negative regulators of cell growth.     

Colonic epithelial expression of ErbB2 is required for postnatal maintenance of the enteric nervous system

We utilized the Cre-LoxP system to establish erbB2 conditional mutant mice in order to investigate the role of erbB2 in postnatal development of the enteric nervous system. The erbB2/nestin-Cre conditional mutants exhibit retarded growth, distended colons, and premature death, resembling human Hirschsprung's disease. Enteric neurons and glia are present at birth in the colon of erbB2/nestin-Cre mutants; however, a marked loss of multiple classes of enteric neurons and glia occurs by 3 weeks of age. Furthermore, we demonstrate that the requirement for erbB2 in maintaining the enteric nervous system is not cell autonomous, but rather erbB2 signaling in the colonic epithelia is required for the postnatal survival of enteric neurons and glia.     

Enteric neuroblasts require the phosphatidylinositol 3-kinase pathway for GDNF-stimulated proliferation

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial-derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest-derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation. We found that GDNF stimulates the phosphorylation of both the PI 3-kinase downstream substrate Akt and the MAP kinase substrate ERK in cultures of immunoaffinity-purified embryonic avian enteric crest-derived cells. The selective PI 3-kinase inhibitor LY-294002 blocked GDNF-stimulated Akt phosphorylation in purified crest cells, and reduced proliferation in cultures of dissociated quail gut. The ERK kinase (MEK) inhibitors PD 98059 and UO126 did not reduce GDNF-stimulated proliferation, although PD 98059 blocked GDNF-stimulated phosphorylation of ERK. We conclude that the PI 3-kinase pathway is necessary for the GDNF-stimulated proliferation of enteric neuroblasts.     

Enteric neuroblasts require the phosphatidylinositol 3‐kinase pathway for GDNF‐stimulated proliferation

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial‐derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest‐derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation. We found that GDNF stimulates the phosphorylation of both the PI 3‐kinase downstream substrate Akt and the MAP kinase substrate ERK in cultures of immunoaffinity‐purified embryonic avian enteric crest‐derived cells. The selective PI 3‐kinase inhibitor LY‐294002 blocked GDNF‐stimulated Akt phosphorylation in purified crest cells, and reduced proliferation in cultures of dissociated quail gut. The ERK kinase (MEK) inhibitors PD 98059 and UO126 did not reduce GDNF‐stimulated proliferation, although PD 98059 blocked GDNF‐stimulated phosphorylation of ERK. We conclude that the PI 3‐kinase pathway is necessary for the GDNF‐stimulated proliferation of enteric neuroblasts. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 306–317, 2001     

Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect

Mice carrying heterozygous mutations in the Sox10 gene display aganglionosis of the colon and represent a model for human Hirschsprung disease. Here, we show that the closely related Sox8 functions as a modifier gene for Sox10-dependent enteric nervous system defects as it increases both penetrance and severity of the defect in Sox10 heterozygous mice despite having no detectable influence on enteric nervous system development on its own. Sox8 exhibits an expression pattern very similar to Sox10 with occurrence in vagal and enteric neural crest cells and later confinement to enteric glia. Loss of Sox8 alleles in Sox10 heterozygous mice impaired colonization of the gut by enteric neural crest cells already at early times. Whereas proliferation, apoptosis, and neuronal differentiation were normal for enteric neural crest cells in the gut of mutant mice, apoptosis was dramatically increased in vagal neural crest cells outside the gut. The defects in enteric nervous system development of mice with Sox10 and Sox8 mutations are therefore likely caused by a reduction of the pool of undifferentiated vagal neural crest cells. Our study suggests that Sox8 and Sox10 are jointly required for the maintenance of these vagal neural crest stem cells.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

The role of enteric glia in gut inflammation

A neuro-glia interaction is part of gut inflammation and essential for the integrity of the bowel. A loss of enteric glia cells (EGCs) led to a fatal haemorrhagic jejuno-ileitis and death in a few days. Although a diminished EGC network is postulated in inflammatory bowel disease and enteric glia pathology is described in Chagas' disease the role of EGCs in the onset of these disease complexes is not definitely clear. Several lines of evidence implicate that the secretion of different factors by enteric glia may be the key for modulating gut homeostasis. As mucosal integrity might be important for remission in Crohn's disease and inflammation of the enteric nervous system is part of the pathology in Chagas' disease, the role of EGCs during gut inflammation could be part of the key to understand these diseases.     

The emergence of neural activity and its role in the development of the enteric nervous system

The enteric nervous system (ENS) is a vital part of the autonomic nervous system that regulates many gastrointestinal functions, including motility and secretion. All neurons and glia of the ENS arise from neural crest-derived cells that migrate into the gastrointestinal tract during embryonic development. It has been known for many years that a subpopulation of the enteric neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development. Recent studies have demonstrated that some enteric neurons exhibit electrical activity from as early as E11.5 in the mouse, with further maturation of activity during embryonic and postnatal development. This article discusses the maturation of electrophysiological and morphological properties of enteric neurons, the formation of synapses and synaptic activity, and the influence of neural activity on ENS development.     

Characterization of a plasma membrane protein present in non-myelin-forming PNS and CNS glia,a subpopulation of PNS neurons,perineurial cells and smooth muscle in adult rats

Summary A plasma membrane protein common to nonmyelin-forming peripheral glia, including non-myelin-forming Schwann cells, satellite cells and enteric glia, is recognized and defined by monoclonal antibody A5E3. It is not detectable immunohistochemically on myelin-forming Schwann cells. The antigen is also present in large amounts on smooth muscle cells and perineurial cells, on some PNS neurons, and at lower levels on astrocytes of adult rat. In neonatal but not adult animals, the antigen is present on skeletal muscle fibres and myoblasts. In immunoblots and immune precipitation experiments on smooth muscle and Schwann cell extracts the antigen is a polypeptide with an apparent molecular weight of 130 kd. In being present in some non-neural tissues, albeit very highly restricted in cell type, this antigen resembles several other cell surface glycoproteins found in large amounts in the nervous system.     

Control of peripheral glial cell proliferation: a comparison of the division rates of enteric glia and Schwann cells and their response to mitogens

The enteric nervous system comprises neurons and a relatively homogeneous population of glial cells, which differ considerably from those found in other parts of the peripheral nervous system and resemble more closely astrocytes from the central nervous system. It provides a simple model system for the study of neuron/glial interactions and glial cell development. In this study the proliferation rates of purified populations of enteric glia and Schwann cells and their response to several mitogens in vitro were compared. Enteric glial cells divided at a much higher rate than Schwann cells in both serum-containing and serum-free media. This difference in their basal proliferation rates was the major difference seen between the two cell types. Both cell populations were stimulated to divide by fibroblast growth factor and glial growth factor but not by epidermal growth factor. Enteric glial cells and Schwann cells proliferated at a greater rate on a basement membrane-like extracellular matrix produced by corneal endothelial cells, laminin, and fibronectin than on poly-L-lysine-coated glass coverslips. The magnitude of stimulation was greater for Schwann cells, presumably due to their lower basal division rates. Like Schwann cells, enteric glial cells were stimulated to divide by two agents which elevate intracellular cAMP, cholera toxin, and dibutyryl cAMP.     

Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia

The vagal neural crest is the origin of majority of neurons and glia that constitute the enteric nervous system, the intrinsic innervation of the gut. We have recently confirmed that a second region of the neuraxis, the sacral neural crest, also contributes to the enteric neuronal and glial populations of both the myenteric and the submucosal plexuses in the chick, caudal to the level of the umbilicus. Results from this previous study showed that sacral neural crest-derived precursors colonised the gut in significant numbers only 4 days after vagal-derived cells had completed their migration along the entire length of the gut. This observation suggested that in order to migrate into the hindgut and differentiate into enteric neurons and glia, sacral neural crest cells may require an interaction with vagal-derived cells or with factors or signalling molecules released by them or their progeny. This interdependence may also explain the inability of sacral neural crest cells to compensate for the lack of ganglia in the terminal hindgut of Hirschsprung's disease in humans or aganglionic megacolon in animals. To investigate the possible interrelationship between sacral and vagal-derived neural crest cells within the hindgut, we mapped the contribution of various vagal neural crest regions to the gut and then ablated appropriate sections of chick vagal neural crest to interrupt the migration of enteric nervous system precursor cells and thus create an aganglionic hindgut model in vivo. In these same ablated animals, the sacral level neural axis was removed and replaced with the equivalent tissue from quail embryos, thus enabling us to document, using cell-specific antibodies, the migration and differentiation of sacral crest-derived cells. Results showed that the vagal neural crest contributed precursors to the enteric nervous system in a regionalised manner. When quail-chick grafts of the neural tube adjacent to somites 1-2 were performed, neural crest cells were found in enteric ganglia throughout the preumbilical gut. These cells were most numerous in the esophagus, sparse in the preumbilical intestine, and absent in the postumbilical gut. When similar grafts adjacent to somites 3-5 or 3-6 were carried out, crest cells were found within enteric ganglia along the entire gut, from the proximal esophagus to the distal colon. Vagal neural crest grafts adjacent to somites 6-7 showed that crest cells from this region were distributed along a caudal-rostral gradient, being most numerous in the hindgut, less so in the intestine, and absent in the proximal foregut. In order to generate aneural hindgut in vivo, it was necessary to ablate the vagal neural crest adjacent to somites 3-6, prior to the 13-somite stage of development. When such ablations were performed, the hindgut, and in some cases also the cecal region, lacked enteric ganglionated plexuses. Sacral neural crest grafting in these vagal neural crest ablated chicks showed that sacral cells migrated along normal, previously described hindgut pathways and formed isolated ganglia containing neurons and glia at the levels of the presumptive myenteric and submucosal plexuses. Comparison between vagal neural crest-ablated and nonablated control animals demonstrated that sacral-derived cells migrated into the gut and differentiated into neurons in higher numbers in the ablated animals than in controls. However, the increase in numbers of sacral neural crest-derived neurons within the hindgut did not appear to be sufficiently high to compensate for the lack of vagal-derived enteric plexuses, as ganglia containing sacral neural crest-derived neurons and glia were small and infrequent. Our findings suggest that the neuronal fate of a relatively fixed subpopulation of sacral neural crest cells may be predetermined as these cells neither require the presence of vagal-derived enteric precursors in order to colonise the hindgut, nor are capable of dramatically altering their proliferation or d     

Interleukin-6 expression and regulation in rat enteric glial cells

As yet, little is known about the function of the glia of the enteric nervous system (ENS), particularly in an immune-stimulated environment. This prompted us to study the potential of cultured enteroglial cells for cytokine synthesis and secretion. Jejunal myenteric plexus preparations from adult rats were enzymatically dissociated, and enteroglial cells were purified by complement-mediated cytolysis and grown in tissue culture. Cultured cells were stimulated with recombinant rat interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, and IL-6 mRNA expression and secretion were assessed using RT-PCR and a bioassay, respectively. Stimulation with TNF-alpha did not affect IL-6 mRNA expression, whereas IL-1beta stimulated IL-6 mRNA and protein synthesis in a time- and concentration-dependent fashion. In contrast, IL-6 significantly and dose-dependently suppressed IL-6 mRNA expression. In summary, we have presented evidence that enteric glial cells are a potential source of IL-6 in the myenteric plexus and that cytokine production by enteric glial cells can be regulated by cytokines. These findings strongly support the contention that enteric glial cells act as immunomodulatory cells in the enteric nervous system.     

Enteric Neuropathy Can Be Induced by High Fat Diet In Vivo and Palmitic Acid Exposure In Vitro

Objective

Obese and/or diabetic patients have elevated levels of free fatty acids and increased susceptibility to gastrointestinal symptoms. Since the enteric nervous system is pivotal in regulating gastrointestinal functions alterations or neuropathy in the enteric neurons are suspected to occur in these conditions. Lipid induced intestinal changes, in particular on enteric neurons, were investigated in vitro and in vivo using primary cell culture and a high fat diet (HFD) mouse model.

Design

Mice were fed normal or HFD for 6 months. Intestines were analyzed for neuronal numbers, remodeling and lipid accumulation. Co-cultures of myenteric neurons, glia and muscle cells from rat small intestine, were treated with palmitic acid (PA) (0 – 10⁻³ M) and / or oleic acid (OA) (0 – 10⁻³ M), with or without modulators of intracellular lipid metabolism. Analyses were by immunocyto- and histochemistry.

Results

HFD caused substantial loss of myenteric neurons, leaving submucous neurons unaffected, and intramuscular lipid accumulation in ileum and colon. PA exposure in vitro resulted in neuronal shrinkage, chromatin condensation and a significant and concentration-dependent decrease in neuronal survival; OA exposure was neuroprotective. Carnitine palmitoyltransferase 1 inhibition, L-carnitine- or alpha lipoic acid supplementation all counteracted PA-induced neuronal loss. PA or OA alone both caused a significant and concentration-dependent loss of muscle cells in vitro. Simultaneous exposure of PA and OA promoted survival of muscle cells and increased intramuscular lipid droplet accumulation. PA exposure transformed glia from a stellate to a rounded phenotype but had no effect on their survival.

Conclusions

HFD and PA exposure are detrimental to myenteric neurons. Present results indicate excessive palmitoylcarnitine formation and exhausted L-carnitine stores leading to energy depletion, attenuated acetylcholine synthesis and oxidative stress to be main mechanisms behind PA-induced neuronal loss.High PA exposure is suggested to be a factor in causing diabetic neuropathy and gastrointestinal dysregulation.     

Glutamate Receptor Subunit Expression in Primary Enteric Glia Cultures

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.     

Development of the enteric nervous system in chick embryonic duodenum in terms of the distribution of tubulin

An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulin-immunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.     

Glutamate receptor subunit expression in primary enteric glia cultures

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.     

Bystander attenuation of neuronal and astrocyte intercellular communication by murine cytomegalovirus infection of glia

Astrocytes are the first cells infected by murine cytomegalovirus (MCMV) in primary cultures of brain. These cells play key roles in intercellular signaling and neuronal development, and they modulate synaptic activity within the nervous system. Using ratiometric fura-2 digital calcium imaging of >8,000 neurons and glia, we found that MCMV-infected astrocytes showed an increase in intracellular basal calcium levels and an enhanced response to neuroactive substances, including glutamate and ATP, and to high potassium levels. Cultured neurons with no sign of MCMV infection showed attenuated synaptic signaling after infection of the underlying astrocyte substrate, and intercellular communication between astrocytes with no sign of infection was reduced by the presence of infected glia. These bystander effects would tend to cause further deterioration of cellular communication in the brain in addition to the problems caused by the loss of directly infected cells.