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.     

Calcium signalling in sensory neurones and peripheral glia in the context of diabetic neuropathies

Peripheral sensory nervous system is comprised of neurones with their axons and neuroglia that includes satellite glial cells in sensory ganglia, myelinating, non-myelinating and perisynaptic Schwann cells. Pathogenesis of peripheral diabetic polyneuropathies is associated with aberrant function of both neurones and glia. Deregulated Ca²⁺ homoeostasis and aberrant Ca²⁺ signalling in neuronal and glial elements contributes to many forms of neuropathology and is fundamental to neurodegenerative diseases. In diabetes both neurones and glia experience metabolic stress and mitochondrial dysfunction which lead to deregulation of Ca²⁺ homeostasis and Ca²⁺ signalling, which in their turn lead to pathological cellular reactions contributing to development of diabetic neuropathies. Molecular cascades responsible for Ca²⁺ homeostasis and signalling, therefore, can be regarded as potential therapeutic targets.     

Different neural crest populations exhibit diverse proliferative behaviors

The rate of proliferation of cells depends on the proportion of cycling cells and the frequency of cell division. Here, we describe in detail methods for quantifying the proliferative behavior of specific cell types in situ, and use the method to examine cell cycle dynamics in two neural crest derivatives—dorsal root ganglia (DRG) using frozen sections, and the enteric nervous system (ENS) using wholemount preparations. In DRG, our data reveal a significant increase in cell cycle length and a decrease in the number of cycling Sox10+ progenitor cells at E12.5–E13.5, which coincides with the commencement of glial cell generation. In the ENS, the vast majority of Sox10+ cells remain proliferative during embryonic development, with only relatively minor changes in cell cycle parameters. Previous studies have identified proliferating cells expressing neuronal markers in the developing ENS; our data suggest that most cells undergoing neuronal differentiation in the developing gut commence expression of neuronal markers during G2 phase of their last division. Combined with previous studies, our findings show that different populations of neural crest‐derived cells show tissue‐specific patterns of proliferation. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 287–301, 2015     

Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

The N‐Myc Downstream‐Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4 ^−/−) CRC models and an indirect co‐culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4 ^−/− ENS cell secretome, which is enriched for Nidogen‐1 (Nid1) and Fibulin‐2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS‐derived Nidogen‐1 and Fibulin‐2 enhance colorectal carcinogenesis.     

Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease

Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret^−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret^−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.     

From neural crest to bowel: Development of the enteric nervous system

The ENS resembles the brain and differs both physiologically and structurally from any other region of the PNS. Recent experiments in which crest cell migration has been studied with DiI, a replication-deficient retrovirus, or antibodies that label cells of neural crest origin, have confirmed that both the avian and mammalian bowel are colonized by émigrés from the sacral as well as the vagal level of the neural crest. Components of the extracellular matrix, such as laminin, may play roles in enteric neural and glial development. The observation that an overabundance of laminin develops in the presumptive aganglionic region of the gut in Is/Is mutant mice and is associated with the inability of crest-derived cells to colonize this region of the bowel has led to the hypothesis that laminin promotes the development of crest-derived cells as enteric neurons. Premature expression of a neuronal phenotype would cause crest-derived cells to cease migrating before they complete the colonization of the gut. The acquisition by crest-derived cells of a nonintegrin, nervespecific, 110 kD laminin-binding protein when they enter the bowel may enable these cells to respond to laminin differently from their pre-enteric migrating predecessors. Crest-derived cells migrating along the vagal pathway to the mammalian gut are transiently catecholaminergic (TC). This phenotype appears to be lost rapidly as the cells enter the bowel and begin to follow their program of terminal differentiation. The appearance and disappearance of TC cells may thus be an example of the effects of the enteric microenvironment on the differentiation of crest-derived cells in situ. Crest-derived cells can be isolated from the enteric microenvironment by immunoselection, a method that takes advantage of the selective expression on the surfaces of crest-derived cells of certain antigens. One neurotrophin, NT-3, promotes the development of enteric neurons and glia in vitro. Because trkC is expressed in the developing and mature gut, it seems likely that NT-3 plays a critical role in the development of the ENS in situ. Although the factors that are responsible for the development of the unique properties of the ENS remain unknown, progress made in understanding enteric neuronal development has recently accelerated. The application of new techniques and recently developed probes suggest that the accelerated pace of discovery in this area can be expected to continue. © 1993 John Wiley & Sons, Inc.     

Retinaldehyde dehydrogenase enzymes regulate colon enteric nervous system structure and function

The enteric nervous system (ENS) forms from the neural crest-derived precursors that colonize the bowel before differentiating into a network of neurons and glia that control intestinal function. Retinoids are essential for normal ENS development, but the role of retinoic acid (RA) metabolism in development remains incompletely understood. Because RA is produced locally in the tissues where it acts by stimulating RAR and RXR receptors, RA signaling during development is absolutely dependent on the rate of RA synthesis and degradation. RA is produced by three different enzymes called retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3) that are all expressed in the developing bowel. To determine the relative importance of these enzymes for ENS development, we analyzed whole mount preparations of adult (8–12-week old) myenteric and submucosal plexus stained with NADPH diaphorase (neurons and neurites), anti-TuJ1 (neurons and neurites), anti-HuC/HuD (neurons), and anti-S100β (glia) in an allelic series of mice with mutations in Raldh1, Raldh2, and Raldh3. We found that Raldh1^−/−, Raldh2^+/−, Raldh3^+/− (R1^KOR2^HetR3^Het) mutant mice had a reduced colon myenteric neuron density, reduced colon myenteric neuron to glia ratio, reduced colon submucosal neuron density, and increased colon myenteric fibers per neuron when compared to the wild type (WT; Raldh1^WT, Raldh2^WT, Raldh3^WT) mice. These defects are unlikely to be due to defective ENS precursor migration since R1^KOR2^HetR3^KO mice had increased enteric neuron progenitor migration into the distal colon compared to WT during development. RALDH mutant mice also have reduced contractility in the colon compared to WT mice. These data suggest that RALDH1, RALDH2 and RALDH3 each contribute to ENS development and function.     

The ephrin receptor EphB2 regulates the connectivity and activity of enteric neurons

Highly organized circuits of enteric neurons are required for the regulation of gastrointestinal functions, such as peristaltism or migrating motor complex. However, the factors and molecular mechanisms that regulate the connectivity of enteric neurons and their assembly into functional neuronal networks are largely unknown. A better understanding of the mechanisms by which neurotrophic factors regulate this enteric neuron circuitry is paramount to understanding enteric nervous system (ENS) physiology. EphB2, a receptor tyrosine kinase, is essential for neuronal connectivity and plasticity in the brain, but so far its presence and function in the ENS remain largely unexplored. Here we report that EphB2 is expressed preferentially by enteric neurons relative to glial cells throughout the gut in rats. We show that in primary enteric neurons, activation of EphB2 by its natural ligand ephrinB2 engages ERK signaling pathways. Long-term activation with ephrinB2 decreases EphB2 expression and reduces molecular and functional connectivity in enteric neurons without affecting neuronal density, ganglionic fiber bundles, or overall neuronal morphology. This is highlighted by a loss of neuronal plasticity markers such as synapsin I, PSD95, and synaptophysin, and a decrease of spontaneous miniature synaptic currents. Together, these data identify a critical role for EphB2 in the ENS and reveal a unique EphB2-mediated molecular program of synapse regulation in enteric neurons.     

Modulation of Mn2+ accumulation in cultured rat neuronal and astroglial cells

The effects of physiological concentrations of K⁺ on Mn²⁺ accumulation were compared in rat glial cells and neurons in culture. Increasing the K⁺ concentration in growth medium increased significantly the Mn²⁺ level of the cultivated cells, with glial cells more affected than neurons. Ethanol markedly increased the Mn²⁺ accumulation within glia but not within neurons while ouabaïn caused inhibition of Mn²⁺ uptake with neurons and glial cells. A modulation of the total protein synthesis by Mn²⁺ and ethanol level in the growth medium was observed with glial cells. These data suggest that the mechanisms involved in Mn²⁺ accumulation in glial cells are different from those present in neurons. Moreover, the results are consistent with the hypothesis that Mn²⁺ plays a regulatory role in glial cell metabolism.     

Voltage-gated calcium channel types in cultured C. elegans CEPsh glial cells

The four cephalic sensilla sheath (CEPsh) glial cells are important for development of the nervous system of Caenorhabditis elegans. Whether these invertebrate glia can generate intracellular Ca²⁺ increases, a hallmark of mammalian glial cell excitability, is not known. To address this issue, we developed a transgenic worm with the specific co-expression of genetically encoded red fluorescent protein and green Ca²⁺ sensor in CEPsh glial cells. This allowed us to identify CEPsh cells in culture and monitor their Ca²⁺ dynamics. We show that CEPsh glial cells, in response to depolarization, generate various intracellular Ca²⁺ increases mediated by voltage-gated Ca²⁺ channels (VGCCs). Using a pharmacological approach, we find that the L-type is the preponderant VGCC type mediating Ca²⁺ dynamics. Additionally, using a genetic approach we demonstrate that mutations in three known VGCC α₁-subunit genes, cca-1, egl-19 and unc-2, can affect Ca²⁺ dynamics of CEPsh glial cells. We suggest that VGCC-mediated Ca²⁺ dynamics in the CEPsh glial cells are complex and display heterogeneity. These findings will aid understanding of how CEPsh glial cells contribute to the operation of the C. elegans nervous system.     

Enteric neurons show a primary cilium

The primary cilium is a non‐motile cilium whose structure is 9+0. It is involved in co‐ordinating cellular signal transduction pathways, developmental processes and tissue homeostasis. Defects in the structure or function of the primary cilium underlie numerous human diseases, collectively termed ciliopathies. The presence of single cilia in the central nervous system (CNS) is well documented, including some choroid plexus cells, neural stem cells, neurons and astrocytes, but the presence of primary cilia in differentiated neurons of the enteric nervous system (ENS) has not yet been described in mammals to the best of our knowledge. The enteric nervous system closely resembles the central nervous system. In fact, the ultrastructure of the ENS is more similar to the CNS ultrastructure than to the rest of the peripheral nervous system. This research work describes for the first time the ultrastructural characteristics of the single cilium in neurons of rat duodenum myenteric plexus, and reviews the cilium function in the CNS to propose the possible role of cilia in the ENS cells.     

Ca<Superscript>2+</Superscript> channel currents of cortical neurons from pure and mixed cultures

Voltage-gated Ca²⁺ channels (VGCCs) are key regulators of many neuronal functions, and involved in multiple central nervous system diseases. In the last 30 years, a large number of injury and disease models have been established based on cultured neurons. Culture with serum develops a mixture of neurons and glial cells, while culture without serum develops pure neurons. Both of these neuronal-culture methods are widely used. However, the properties of Ca²⁺ currents in neurons from these two cultures have not been compared. In this study, we cultured rat cortical neurons in serum-containing or -free medium and then recorded the Ca²⁺ channel currents using patch-clamp technique. Our results showed that there were significant differences in the amplitude and activation properties of whole-cell Ca²⁺ channel currents, and of non-L-type Ca²⁺ channel currents between the neurons from these two culture systems. Our data suggested that the difference of whole-cell Ca²⁺ currents may result from the differences in non-L-type currents. Understanding of these properties will considerably advance studies of VGCCs in neurons from pure or mixed culture.     

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.     

Nitric oxide synthase immunoreactivity and NADPH-d histochemistry in the enteric nervous system of Sarda breed sheep with different PrP genotypes in whole-mount and cryostat preparations.

Until now, significant differences in the neurochemical pattern of enteric neurons have been demonstrated in all species studied; however, some strong similarities also occur across species, such as the occurrence of nitric oxide synthase immunoreactivity (NOS-IR) in inhibitory motor neurons to muscle. In consideration of the insufficient data regarding the enteric nervous system (ENS) of sheep, we investigated the myenteric plexus and submucosal plexus of the ovine ileum. Since the pivotal role of the ENS in the early pathogenesis of sheep scrapie, the "prototype" of prion diseases, has been suggested, we have focused our observations also on the host's PrP genotype. We have studied the morphology and distribution of NOS-IR neurons and their relationships with the enteric glia in whole-mount preparations and in cryostat sections. NOS-IR neurons, always encircled by glial processes, were located in both plexuses. Many NOS-IR fibers were seen in the circular muscle layer, in the submucosa, and in the mucosa. In the submucosa they were close to the lymphoid tissue. No differences in the distribution and percentage of NOS-IR fibers and neurons were observed among sheep carrying different PrP genotype, thus making unlikely their contribution in the determinism of susceptibility/resistance to scrapie infection.     

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.     

The Local Control of Cytosolic Ca2+ as a Propagator of CNS Communication—Integration of Mitochondrial Transport Mechanisms and Cellular Responses

Ca²⁺ signals propagate in wave form along individual cells of the central nervous system(CNS) and through networks of connected cells of neuronal and multiple glial cell types. Inorder for wave fronts to convey information, signaling mechanisms are required that allowwaves to propagate reproducibly and without decrement in signal strength over long distances.CNS Ca²⁺ waves are under specific integrated local control, made possible by interactions atlocal subcellular microdomains between endoplasmic reticulum and mitochondria. Activemitochondria located near the mouth of inositol trisphosphate receptor (InsP₃R) channel clustersin glia take up Ca²⁺, which may prevent a buildup of Ca²⁺ around the InsP₃R channel, therebydecreasing the rate of Ca²⁺-induced receptor inactivation, and prolonging channel open time.Mitochondria may amplify InsP;i3-dependent Ca2;pl signals by a transient permeability transitionin response to Ca²⁺ uptake into the mitochondrion. Other evidence suggests privileged accessinto mitochondria for Ca²⁺ entering neurons by glutamatergic receptor channels. This enablesspecific signal modulation as the Ca²⁺ wave is propagated into neurons, such that mitochondrialocated close to glutamate channels can prolong the neuronal cytosolic response time bysuccessive uptake and release of Ca²⁺. Disruption of mitochondrial function deregulates theability of CNS-derived cells to undergo normal Ca²⁺ signaling and wave propagation.     

Phactr4: A new integrin modulator required for directional migration of enteric neural crest cells

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

Calcium signals activated by ghrelin and D-Lys-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys³-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca²⁺]_i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca²⁺ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys³-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca²⁺ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys³-GHRP-6 ghrelin antagonist features ghrelin independent activities.     

The dual face of connexin-based astroglial Ca communication: A key player in brain physiology and a prime target in pathology

For decades, studies have been focusing on the neuronal abnormalities that accompany neurodegenerative disorders. Yet, glial cells are emerging as important players in numerous neurological diseases. Astrocytes, the main type of glia in the central nervous system , form extensive networks that physically and functionally connect neuronal synapses with cerebral blood vessels. Normal brain functioning strictly depends on highly specialized cellular cross-talk between these different partners to which Ca^2 +, as a signaling ion, largely contributes. Altered intracellular Ca^2 + levels are associated with neurodegenerative disorders and play a crucial role in the glial responses to injury. Intracellular Ca^2 + increases in single astrocytes can be propagated toward neighboring cells as intercellular Ca^2 + waves, thereby recruiting a larger group of cells. Intercellular Ca²⁺ wave propagation depends on two, parallel, connexin (Cx) channel-based mechanisms: i) the diffusion of inositol 1,4,5-trisphosphate through gap junction channels that directly connect the cytoplasm of neighboring cells, and ii) the release of paracrine messengers such as glutamate and ATP through hemichannels (‘half of a gap junction channel’). This review gives an overview of the current knowledge on Cx-mediated Ca^2 + communication among astrocytes as well as between astrocytes and other brain cell types in physiology and pathology, with a focus on the processes of neurodegeneration and reactive gliosis. Research on Cx-mediated astroglial Ca^2 + communication may ultimately shed light on the development of targeted therapies for neurodegenerative disorders in which astrocytes participate. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.