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.     

Immunohistochemical detection of [corrected] TrkB in the enteric nervous system of the small intestine in pigeon (Columba livia)

The presence and cell localization of TrkB, the main receptor for the neurotrophins (NTs), was investigated immunohistochemically in the small intestine of adult pigeons, with special reference to the enteric nervous system (ENS). Several neuronal (neurofilament proteins and PGP 9.5) and glial cell (S100 protein) markers were studied in parallel. TrkB immunoreactivity (TrkB-IR) was found to be restricted to immunohistochemically-identified glial cells present in the enteric plexuses, and to Schwann cells forming the perivascular plexus. Also, TrkB-IR was detected in enterochromaffin cells and in unidentified dendritic cells within the gut-associated lymphoid tissue. The present results demonstrate that as for mammals, TrkB in the ENS is restricted to the glial cells. The possible function of the TrkB ligands, however, remains to be established.     

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.     

Prospective identification and culture of rat enteric neural stem cells (ENSCs)

Hirschprung’s disease (HD), a very common congenital abnormality in children, occurs mainly due to the congenital developmental defect of the enteric nervous system. The absence of enteric ganglia from the distal gut due to deletion in gut colonization by neural crest progenitor cells may lead to HD. The capacity to identify and isolate the enteric neuronal precursor cells from developing and mature tissues would enable the development of cell replacement therapies for HD. However, a mature method to culture these cells is a challenge. The present study aimed to propose a method to culture enteric neural stem cells (ENSCs) from the DsRed transgenic fetal rat gut. The culture medium used contained 15 % chicken embryo extract, basic fibroblast growth factor, and epidermal growth factor. ENSCs were cultured from embryonic day 18 in DsRed transgenic rat. Under inverted microscope and fluorescence staining, ENSCs proliferated to form small cell clusters on the second day of culture. The neurospheres-like structure were suspended in the medium, and there were some filaments between the adherent cells from day 3 to day 6 of the culture. The neurospheres were formed by ENSCs on day 8 of the culture. Network-like connections were formed between the adherent cells and differentiated cells after adding 10 % FBS. The differentiated cells were positive for neurofilament and glial fibrillary acidic protein antibodies. The present study established a method to isolate and culture ENSCs from E18 DsRed transgenic rats in the terminal stage of embryonic development. This study would offer a way to obtain plenty of cells for the future research on the transplantation of HD.     

Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system.

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.     

MEGF9: a novel transmembrane protein with a strong and developmentally regulated expression in the nervous system

MEGF9 [multiple EGF (epidermal growth factor)-like-domains 9], a novel transmembrane protein with multiple EGF-like repeats, is predominantly expressed in the developing and adult CNS (central nervous system) and PNS (peripheral nervous system). The domain structure of MEGF9 consists of an N-terminal region with several potential O-glycosylation sites followed by five EGF-like domains, which are highly homologous with the short arms of laminins. Following one single pass transmembrane domain, a highly conserved short intracellular domain with potential phosphorylation sites is present. The protein was recombinantly expressed and characterized as a tissue component. To study the expression pattern further, immunohistochemistry was performed and staining was detected in Purkinje cells of the cerebellum and in glial cells of the PNS. Additional expression was observed in the epidermal layer of skin, papillae of the tongue and the epithelium of the gastrointestinal tract. By immunoelectron microscopy, MEGF9 was detected in glial cells of the sciatic nerve facing the basement membrane. MEGF9 represents a novel putative receptor, expressed in neuronal and non-neuronal tissues, that is regulated during development and could function as a guidance or signalling molecule.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Localization and actions of transforming growth factor-beta s in the embryonic nervous system.

We present evidence for unique localization and specific biological activities for transforming growth factor-beta s (TGF-beta s) 2 and 3, as compared to TGF-beta 1, in the nervous system of the 12-18 day mouse embryo. Each TGF-beta isoform was localized immunohistochemically by specific antibodies raised to peptides corresponding to unique sequences in the respective TGF-beta proteins. Staining for TGF-beta 1 was principally in the meninges, while TGF-beta s 2 and 3 co-localized in neuronal perikarya and axons, as well as in radial glial cells. In the central nervous system, staining was most prominent in zones where neuronal differentiation occurs and less intense in zones of active proliferation, while in the peripheral nervous system, many nerve fibers as well as their cell bodies were strongly immunoreactive for TGF-beta s 2 and 3. Functionally, we have also found that in the presence of an extract of chick eye tissue, TGF-beta s 2 and 3 inhibit survival of cultured embryonic chick ciliary ganglionic neurons in a dose-dependent fashion; TGF-beta 1 shows no inhibitory effects. Our data suggest that TGF-beta s 2 and 3 may play a role in regulation of neuronal migration and differentiation, as well as in glial cell proliferation and differentiation.     

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.     

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.     

Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures

Cultures of dissociated foetal and postnatal mouse gut gave rise to neurosphere-like bodies, which contained large numbers of mature neurons and glial cells. In addition to differentiated cells, neurosphere-like bodies included proliferating progenitors which, when cultured at clonal densities, gave rise to colonies containing many of the neuronal subtypes and glial cells present in the mammalian enteric nervous system. These progenitors were also capable of colonising wild-type and aganglionic gut in organ culture and had the potential to generate differentiated progeny that localised within the intrinsic ganglionic plexus. Similar progenitors were also derived from the normoganglionic small intestine of mice with colonic aganglionosis. Our findings establish the feasibility of expanding and isolating early progenitors of the enteric nervous system based on their ability to form distinct neurogenic and gliogenic structures in culture. Furthermore, these experiments provide the rationale for the development of novel approaches to the treatment of congenital megacolon (Hirschsprung's disease) based on the colonisation of the aganglionic gut with progenitors derived from normoganglionic bowel segments.     

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     

Immunocytochemical localization of carbonic anhydrase in myelinated fibers in peripheral nerves of rat and mouse

Rat sciatic nerve, spinal root, and cranial nerve were immunostained with an antibody against rat brain carbonic anhydrase II (ca), to determine the localization of ca in the rat peripheral nervous system (PNS). Similar methods were applied to mouse nerves to see if that antigen could be detected in the PNS of this species. In rat nerves, intense immunostaining was observed in the axoplasm of many of the myelinated fibers, whereas others were stained less intensely or were negative. A heterogeneous pattern of immunostaining was also found in neuronal perikarya within the ganglia, and in some regions of the ganglia ca immunostaining was found in putative satellite cells and their processes. Ca in rat PNS therefore appears to occur at both neuronal and glial sites, whereas it is exclusively glial in the CNS. In longitudinal sections of some fibers within rat nerves, ca immunostaining could be detected at the inner boundaries of the myelin sheaths. In mouse nerves, axoplasmic staining was observed but it was fainter than in rat nerves. Interspecies differences were most obvious in the dorsal columns of the spinal cord. In rat, intensely stained axons proceeded through the roots into the gracilis or cuneate and often into the gray matter. In mouse, there was much less immunostaining of axons but more intense ca immunostaining in CNS myelin than in the CNS myelin in the rat cord. The implications concerning putative functions of ca in the rodent nervous system are discussed.     

Localization of 4.1 related proteins in cerebellar neurons

Localization of 4.1 related proteins in neurons was studied with immunofluorescence microscopy and with immunoelectron microscopy on ultrathin cryosections. In rat cerebellum, 4.1 immunoreactive proteins were demonstrated in Purkinje cell bodies, dendrites and other neurons in the cerebellar cortex. Some glial cells showed staining, but no labeling was found in myelinated axons of the white matter and of the glomeruli in the granule cell layer. At the ultrastructural level, the 4.1 related proteins were localized mainly in the cytoplasmic matrix, while some labeling was found underneath the plasma membrane. To determine whether 4.1 related proteins in neuronal cytoplasm exist as part of the cytoskeleton or not, PC12 cells cultured in the presence of nerve growth factor were stained with the anti-4.1 antibody. Since cytoplasmic staining was retained after detergent treatment, the 4.1 related proteins seem to exist as a component of the neural cell cytoskeleton. Localization of 4.1 related proteins during the postnatal development of the cerebellum was also studied. In Purkinje cells, localization of 4.1 related proteins changed according to the stages of the postnatal development. The present data suggest that 4.1 related proteins in neurons localized mainly in the cytoplasm and may play some role in organizing cytoskeletal networks in the cytomatrix. Their distribution is developmentally regulated in some neurons, possibly in relationship to their maturation in the cytoskeleton.     

Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.     

Development of the enteric nervous system

The mature enteric nervous system (ENS) is characterized by a degree of neuronal phenotypic diversity and independence of central nervous system control unequaled by any other region of the peripheral nervous system. Studies that have utilized the immunocytochemical demonstration of neurofilament protein and explanation of primordial gut with subsequent growth in culture have indicated that the neural crest precursors of enteric neurons are already committed to the neuronal lineage when they colonize the bowel; however, neuronal phenotypic expression occurs within the gut itself. It is likely that precursors able to give rise to each type of neuron found in the mature ENS are present among the earliest neural crest émigrés to reach the bowel. In mice a proximodistal wave of neuronal phenotypic expression occurs that does not appear to reflect the descent of neuronal precursors. This observation, the known plasticity of developing neural crest-derived neurons, and the demonstration of a persistent population of proliferating neuroblasts in the gut raise the possibility that enteric neuronal phenotypic expression is influenced by the enteric microenvironment.     

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.     

Immunocytochemical localization of NA+, K+-ATPase in primary cultures of rat retina

Conditions have been established which allow growth of embryonic rat retinal cells in dissociated cell culture for up to one month. Na+, K+-ATPase localization was studied in both neuronal and mixed neuronal-glial (flat cell) cultures. High Na+, K+-ATPase-like-immunoreactivity was associated with plasma membranes of neuronal cell bodies and their processes. Markedly lower immunoreactivity was found in the underlying flat cells in mixed cultures. Staining was generally uniform over perikaryal plasma membranes and showed a bead-like appearance in neuronal processes, supporting previous studies in brain tissue which used histocytochemical procedures specific for the Na+, K+-ATPase. This system should be useful for examining distribution of the enzyme in developing nerve and glial cells and may help to resolve questions regarding Na+-K+ homeostasis by neurons and glia.     

Ca(2+)-ATPase and Mg(2+)-ATPase in Aplysia glial and interstitial cells: an EM cytochemical study.

The localization of Ca(2+)- and Mg(2+)-ATPases was determined in Aplysia central and peripheral nervous system, using an electron microscopic cytochemical method. The enzyme activity appeared localized to the membrane of glial granules (gliagrana), particularly in the peripheral nervous system of the esophagus, and on the plasma membrane of central glial cells adjacent to neuronal cell bodies. No calcium- and/or magnesium-ATPase activity was detectable on the plasma membrane of glial cells surrounding nerve axons in the pleuro-visceral connectives. These findings are discussed along two main lines: (a) the calcium-ATPase of the gliagrana coincides with a high intragranular calcium and/or proton concentration; and (b) the presence of a calcium-ATPase activity at the glio-neuronal interface around the neuronal cell bodies coincides with the use of calcium ions as charge carriers of the action potential, and its absence at the level of the axon with the concurrent functional use of sodium ions.