Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system

Enteric glial cells are vital for the autonomic control of gastrointestinal homeostasis by the enteric nervous system. Several different functions have been assigned to enteric glial cells but whether these are performed by specialized subtypes with a distinctive phenotype and function remains elusive. We used Mosaic Analysis with Double Markers and inducible lineage tracing to characterize the morphology and dynamic molecular marker expression of enteric GLIA in the myenteric plexus. Functional analysis in individually identified enteric glia was performed by Ca²⁺ imaging. Our experiments have identified four morphologically distinct subpopulations of enteric glia in the gastrointestinal tract of adult mice. Marker expression analysis showed that the majority of glia in the myenteric plexus co‐express glial fibrillary acidic protein (GFAP), S100β, and Sox10. However, a considerable fraction (up to 80%) of glia outside the myenteric ganglia, did not label for these markers. Lineage tracing experiments suggest that these alternative combinations of markers reflect dynamic gene regulation rather than lineage restrictions. At the functional level, the three myenteric glia subtypes can be distinguished by their differential response to adenosine triphosphate. Together, our studies reveal extensive heterogeneity and phenotypic plasticity of enteric glial cells and set a framework for further investigations aimed at deciphering their role in digestive function and disease. GLIA 2015;63:229–241     

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice

Abstract  The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro–glia interaction. The aim was to investigate neuro–glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca²⁺-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia–neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca²⁺-response to either depolarization (high K⁺) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPβs. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPβs. Activation of neuronal cells (DMPP, K⁺) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.     

Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin‐1β from enteric glial cells

Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron–glia interaction in vitro. We examined the increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the release of interleukin‐1β (IL‐1β) or prostaglandin E₂ (PGE₂) and COX‐2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK‐induced [Ca²⁺]_i increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca²⁺]_i. The effects of LPS were blocked by pretreatment with an interleukin‐1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL‐1β from enteric glial cells. The augmenting effects of IL‐1β on the BK‐induced neural [Ca²⁺]_i increase and PGE₂ release from enteric glial cells were abolished by a phospholipase A₂ (PLA₂) inhibitor and a COX inhibitor, and partly suppressed by a COX‐2 inhibitor. IL‐1β up‐regulated the COX‐2 expression in enteric glial cells. LPS promotes IL‐1β secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE₂ release via glial PLA₂ and COX‐2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease. © 2009 Wiley‐Liss, Inc.     

Amelioration of enteric neuropathology in a mouse model of Niemann‐Pick C by Npc1 expression in enteric glia

Niemann‐Pick C (NPC) disease is an autosomal recessive, lethal, neurodegenerative disorder caused by mutations in NPC1. By using the glial fibrillary acidic protein (GFAP) promoter, we demonstrated previously that astrocyte‐specific expression of Npc1 decreased neuronal storage of cholesterol in Npc1^?/? mice; reduced numbers of axonal spheroids; and produced less degeneration of neurons, reactive astrocytes, and loss of myelin tracts in the central nervous system. GFAP‐Npc1, Npc1^?/? mice exhibited markedly enhanced survival, and death was not associated with the severe terminal weight loss observed in Npc1^?/? mice. Intestinal transit is delayed in Npc1^?/? mice but is normal in GFAP‐NPC1, Npc1^?/? until late in the course of their disease. Because glia play an important role in the enteric nervous system, we studied morphology and cholesterol content of intestines from Npc1^?/? mice and examined the effect of GFAP‐promoted restoration of Npc1 in enteric glia. Although the number of neurons was not altered, the total amount of cholesterol stored in the small intestine was decreased, as were the number of neurons with inclusions and the number of inclusions per neuron. We conclude that expression of Npc1 by enteric glial cells can ameliorate the enteric neuropathology, and we speculate that dysfunction of the enteric nervous system contributes to the retarded intestinal transit, weight loss, and demise of Npc1^?/? mice. © 2009 Wiley‐Liss, Inc.     

The relationship between glial distortion and neuronal changes following intestinal ischemia and reperfusion

Background Damage to mucosal epithelial cells, muscle cells and enteric neurons has been extensively studied following intestinal ischemia and reperfusion (I/R). Interestingly, the effects of intestinal I/R on enteric glia remains unexplored, despite knowledge that glia contribute to neuronal maintenance. Here, we describe structural damage to enteric glia and associated changes in distribution and immunoreactivity of the neuronal protein Hu. Methods The mouse small intestine was made ischemic for 3 h and reperfused from 1 to 12 h. Immunohistochemical localisation of glial fibrillary acidic protein (GFAP), Hu and TUNEL were used to evaluate changes. Key Results At all time points glial cells became distorted, which was evident by their altered GFAP immunoreactivity, including an unusual appearance of bright perinuclear GFAP staining and the presence of GFAP globules. The numbers of neurons per ganglion area were significantly fewer in ganglia that contained distorted glia when compared with ganglia that contained glia of normal appearance. The distribution of Hu immunoreactivity was altered at all reperfusion time points. The presence of vacuoles and Hu granules in neurons was evident and an increase in nuclear Hu, relative to cytoplasmic Hu, was observed in ganglia that contained both normal and distorted glial cells. A number of neurons appeared to lose their Hu immunoreactivity, most noticeably in ganglia that contained distorted glial cells. TUNEL reaction occurred in a minority of glial cells and neurons. Conclusions & Inferences Structural damage to gliofilaments occurs following I/R and may be associated with damage to neighboring neurons.     

Axons and astrocytes release ATP and glutamate to evoke calcium signals in NG2‐glia

NG2‐glia are an abundant population of cells in the adult CNS that make up a novel glial cell type. Here, we have examined calcium signals in NG2‐glia identified by expression of the fluorescent protein DsRed under the control of the NG2 promoter in the white matter of the mouse optic nerve. We focused on mice aged postnatal day (P)12–16, after the main period of oligodendrocyte generation. Using fluo‐4 and fura‐2 calcium imaging in isolated intact nerves, we show that glutamate and ATP evoke Ca²⁺ signals in NG2‐glia in situ, acting on AMPA‐type glutamate receptors and P2Y₁ and P2X₇ purine receptors; NMDA evoked a weak Ca²⁺ signal in a small proportion of NG2‐glia. We show that axonal action potentials and mechanical stimulation of astrocytes effect the release of glutamate and ATP to act on NG2‐glia; ATP alone evokes robust Ca²⁺ signals, whereas glutamate did not unless AMPA receptor desensitization was blocked with cyclothiazide. We identify the precise contacts that NG2‐glia form with axons at nodes of Ranvier, and the intricate bipartite sheaths formed between the processes of NG2‐glia and astrocytes. In addition, we provide evidence that NG2‐glia express synaptophysin, indicating they have mechanisms for transmitting as well as receiving signals. This study places NG2‐glia within a neuron‐glial network, and identifies roles for glutamate and ATP in communication with astrocytes as well as axons. © 2009 Wiley‐Liss, Inc.     

Intercellular calcium waves in cultured enteric glia from neonatal guinea pig

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura-2 AM microfluorimetry. Single-cell stimulation was performed with a micromanipulator-driven glass pipette. Data were expressed as mean +/- SEM and analyzed by Student's t-test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% +/- 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 microM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 microM) significantly decreased the percentage of cells responding to mechanical stimulation (6% +/- 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% +/- 13% vs. 60% +/- 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles.     

Prolonged IL‐1β exposure alters neurotransmitter and electrically induced Ca2+ responses in the myenteric plexus

Background Infection and inflammatory diseases of the gut results in profound changes of intestinal motor function. Acute administration of the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) was shown to have excitatory and neuromodulatory roles in the myenteric plexus. Here we aimed to study the effect of prolonged IL‐1β incubation on the response of myenteric neurones to different stimuli. Methods Longitudinal muscle myenteric plexus preparations (LMMP’s) of the guinea pig jejunum were incubated for 24 h in medium with or without IL‐1β. After loading with Fluo‐4, calcium imaging was used to visualize activation of neurones. The response to application of serotonin (5‐HT), substance P (SP) and ATP or to electrical fibre tract stimulation (eFTS) was tested. Expression of nNOS, HuD, calbindin and calretinin was compared by immunohistochemistry. Key Results IL‐1β concentration‐dependently influenced the neuronal responsiveness and duration of the [Ca²⁺]_i rises to 5‐HT and ATP, while it also affected the Ca²⁺‐transient amplitudes induced by 5‐HT, ATP and SP. Ca²⁺‐transients in response to eFTS were observed in significantly more neurones per ganglion after IL‐1β (10^?10 and 10^?11 mol L^?1). Peak [Ca²⁺]_i rise after eFTS was concentration‐dependently decreased by IL‐1β. The duration of the [Ca²⁺]_i rise after eFTS was prolonged after IL‐1β 10^?12 mol L^?1. IL‐1β (10^?9 mol L^?1) incubation did not affect the number of nNOS, calretinin and calbindin expressing neurones, nor did it induce neuronal loss (HuD). Conclusions & Inferences In this study, IL‐1β differentially modulates the neuronal response to eFTS and neurotransmitter application in the myenteric plexus of guinea pigs. This cytokine could be implicated in the motility disturbances observed during gastrointestinal inflammation.     

Polarized distribution of AMPA,but not GABAA,receptors in radial glia‐like cells of the adult dentate gyrus

Glial fibrillary acidic protein (GFAP)‐positive astrocytes with radial processes [radial glia (RG)‐like cells] in the postnatal dentate gyrus share many of the characteristics of embryonic radial glia and appear to act as precursor cells for adult dentate neurogenesis, a process important for pattern separation and hippocampus‐dependent learning. Although much work has delineated the mechanisms underlying activity‐neurogenesis coupling via gamma‐amino butyric acid (GABA)ergic neurotransmission on GFAP‐negative transient‐amplifying cells and neuroblasts, little is known regarding the effects of neurotransmitters on RG‐like cells. Conflicting evidence exists for both GABA and glutamate receptors on these cells. Here, using GFAP reporter mice, we show that the somatic membrane of RG‐like cells carries GABA_A receptors and glutamate transporters but not ionotropic glutamate receptors, whereas 2‐amino‐3‐(hydroxyl‐5‐methylisoxazole‐4‐yl) propionic acid (AMPA) and GABA_A receptors are expressed on the processes of these cells. Almost all RG‐like cells expressed the GluA2 subunit, which restricts the Ca²⁺ permeability of AMPA receptors. The glial GABA_A receptors mainly comprised α2/α4, β1, and γ1/γ3. The selective presence of AMPA receptors on the radial processes may be important for sensing and responding to local activity‐driven glutamate release and supports the concept that RG‐like astrocytes are composed of functional and structural domains.     

Calcium dependence of spontaneous neurotransmitter release

Spontaneous release of neurotransmitters is regulated by extracellular [Ca²⁺] and intracellular [Ca²⁺]. Curiously, some of the mechanisms of Ca²⁺ signaling at central synapses are different at excitatory and inhibitory synapses. While the stochastic activity of voltage‐activated Ca²⁺ channels triggers a majority of spontaneous release at inhibitory synapses, this is not the case at excitatory nerve terminals. Ca²⁺ release from intracellular stores regulates spontaneous release at excitatory and inhibitory terminals, as do agonists of the Ca²⁺‐sensing receptor. Molecular machinery triggering spontaneous vesicle fusion may differ from that underlying evoked release and may be one of the sources of heterogeneity in release mechanisms.     

Sensory response in host and engrafted astrocytes of adult brain in Vivo

Rapid advances in Ca²⁺ imaging techniques enable us to simultaneously monitor the activities of hundreds of astrocytes in the intact brain, thus providing a powerful tool for understanding the functions of both host and engrafted astrocytes in sensory processing in vivo. These techniques include both improved Ca²⁺ indicators and advanced optical recording methods. Astrocytes in multiple cortical and sub‐cortical areas are able to respond to the corresponding sensory modalities. These sensory stimuli produce astrocytic Ca²⁺ responses through different cellular mechanisms. In addition, it has been suggested that astrocytic gene deficiencies in various sensory systems cause impairments in sensory circuits and cognition. Therefore, glial transplantation would be a potentially interesting approach for the cell‐based therapy for glia‐related disorders. There are multiple cell sources for glial transplantation, including neural stem cells, glial progenitors, and pluripotent stem cells. Both in vitro and in vivo studies have shown that engrafted astrocytes derived from these cell sources are capable of responding to sensory stimulation by elevating the intracellular Ca²⁺ concentration. These results indicate that engrafted astrocytes not only morphologically but also functionally integrate into the host neural network. Until now, many animal studies have proven that glial transplantation would be a good choice for treating multiple glial disorders. Together, these studies on the sensory responses of host and engrafted astrocytes have provided us a novel perspective in both neuron‐glia circuit functions and future treatment strategies for glial disorders.     

Plasmalemmal and mitochondrial Na+‐Ca2+ exchange in neuroglia

In the absence of the electrical signaling for which neurons are so highly specialized, GLIA rely on the slow propagation of ionic signals to mediate network events such as Ca²⁺ and Na⁺ waves. Glia differ from neurons in another important way, they are replete with a high density of ionic‐transport proteins that are essential for them to fulfil their basic functions as guardians of the intra and extra‐cellular milieux. Both the signaling and the homeostatic properties of glial cells are therefore particularly dependent upon the regulation of the two principle physiological metal cations, Ca²⁺ and Na⁺. For both ions, glia express high‐affinity/low capacity ATP‐fuelled pumps that can rapidly move small numbers of ions against an electro‐chemical gradient. For both Ca²⁺ and Na⁺ regulation, a single transporter family, the Na⁺‐Ca²⁺ exchanger (NCX), is used to maintain cellular ion homeostasis over the longer term and under conditions of prolonged or acute ionic dysregulation in astrocytes, oligodendroglia and microglia. Our understanding of glial NCX, both plasmalemmal and mitochondrial, is undergoing the kind of transformation that our understanding of glial cells, in general, has undergone in recent decades. These exchange proteins are becoming increasingly recognized for their essential roles in intracellular homeostasis while their signaling functions are starting to come to light. This review summarizes these key aspects and highlights the many areas where work has yet to begin in this rapidly evolving field. GLIA 2016;64:1646–1654     

Enteric glial cells: New players in Parkinson's disease?

Lewy pathology has been described in neurons of the enteric nervous system in nearly all Parkinson's disease (PD) patients at autopsy. The enteric nervous system not only contains a variety of functionally distinct enteric neurons but also harbors a prominent component of glial cells, the so‐called enteric glial cells, which, like astrocytes of the central nervous system, contribute to support, protect, and maintain the neural network. A growing body of evidence supports a role for enteric glial cells in the pathophysiology of gastrointestinal disorders such as inflammatory bowel disease and chronic constipation. We have recently shown that enteric glial cell dysfunction occurs in PD. In the present review, we discuss the possible implications of enteric glia in PD‐related gut dysfunction as well as in disease initiation and development. © 2014 International Parkinson and Movement Disorder Society     

Glial fibrillary acidic polypeptides in peripheral glia. Molecular weight, heterogeneity and distribution

Glial fibrillary acidic (GFA) polypeptides are present in major categories of rat peripheral glia including non-myelin-forming Schwann cells, enteric glia and some satellite cells. They can be detected both immunochemically and immunohistochemically. The immunoreactivity is associated with a polypeptide which has an MW of 49 000, indistinguishable from that of glial fibrillary acidic protein (GFAP) from rat brain. In spite of this, the GFA polypeptides found in the peripheral nervous system and central nervous system are not identical since they can be distinguished both immunohistochemically and immunochemically by a monoclonal GFAP antibody which recognizes GFAP in astrocytes and some enteric glia, but not GFAP in non-myelin-forming Schwann cells, satellite cells and many enteric glia. GFA-related molecules can also be detected in human Schwann cells by immunofluorescence. The results suggest, however, that the glial filament polypeptides of peripheral glia and astrocytes are less closely related in the human than in the rat. The glial distribution of GFAP is closely paralleled by 2 cell surface proteins, Ran-2 and A5E3 antigen. Although GFAP, Ran-2 and A5E3 are individually expressed by diverse cell types, the phenotype GFAP+, Ran-2+, A5E3+ defines a narrow group including only non-myelin-forming Schwann cells, enteric glia and astrocytes. These observations suggest that the non-myelin-forming cells of the central and peripheral nervous system may share some common functions.     

Fructose consumption impairs serotonergic signaling in the murine enteric nervous system

The intake of free fructose has increased substantially since the development of high‐fructose corn syrup. This has not only been associated with metabolic disorders but recent evidence also indicates that chronic fructose consumption can affect neuronal and cognitive function. In this study we investigated the effects of fructose consumption on serotonergic signaling and neuronal activity in the mouse submucous plexus. Male mice were put on a control or fructose (23% solution) diet for 6 weeks or were assigned to a recovery group that received normal water (2 weeks) after 4 weeks of fructose. At the end of the diet, gene expressions and enteric neuronal activity, after depolarization with high K⁺ and 5‐HT, were measured using Ca²⁺ imaging and RT‐qPCR, respectively. Even in the lack of gain weight and the absence of changes in duodenal permeability, the total number of 5‐HT‐responding neurons and the depolarization and 5‐HT‐evoked Ca²⁺ amplitudes were significantly lower after fructose consumption. Expression of synaptobrevin Ca_V2.1 and Ca_V2.2 mRNA did not differ after fructose intake; however, Ca_V2.1 mRNA levels were significantly higher in the recovery animals. SERT mRNA concentration, isolated from submucosal plexus containing mucosal epithelium, was significantly decreased after fructose consumption. Chronic fructose consumption impairs serotonergic signaling in the mouse submucous plexus, prior to weight gain and detectable intestinal permeability problems.     

Two modes of enteric gliotransmission differentially affect gut physiology

Enteric glia (EG) in the enteric nervous system can modulate neuronally regulated gut functions. Using molecular genetics, we assessed the effects that molecular entities expressed in EG and otherwise mediating two distinct mechanisms of gliotransmitter release, connexin 43 (Cx43) hemichannel vs. Ca²⁺‐dependent exocytosis, have on gut function. The expression of mutated Cx43^G138R (which favors hemichannel, as opposed to gap‐junctional activity) in EG increased gut motility in vivo, while a knock‐down of Cx43 in EG resulted in the reduction of gut motility. However, inhibition of Ca²⁺‐dependent exocytosis in EG did not affect gut motility in vivo; rather, it increased the fecal pellet fluid content. Hampering either Cx43 expression or Ca²⁺‐dependent exocytosis in EG had an effect on colonic migrating motor complexes, mainly decreasing frequency and velocity of contractions ex vivo. Thus, EG can differentially modulate gut reflexes using the above two distinct mechanisms of gliotransmission.     

Nestin‐expressing cells in the gut give rise to enteric neurons and glial cells

Background Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. Although NSCs have been isolated from intestinal musclularis, their presence in mucosa has not been well described. Mucosa‐derived NSCs are accessible endoscopically and could be used autologously. Brain‐derived Nestin‐positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa‐derived NSCs, determine their relationship to Nestin‐expressing cells and to demonstrate their capacity to produce neuroglial networks in vitro and in vivo. Methods Neurospheres were generated from periventricular brain, colonic muscularis (Musc), and mucosa–submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin‐GFP). Neuronal stem cells were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed using immunohistochemistry using glial and neuronal markers. Brain and gut‐derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. Key Results Musc‐ and MSM‐derived neurospheres expressed Nestin and gave rise to cells of neuronal, glial, and mesenchymal lineage. Although Nestin expression in tissue was mostly limited to glia co‐labelled with glial fibrillary acid protein (GFAP), neurosphere‐derived neurons and glia both expressed Nestin in vitro, suggesting that Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover, following transplantation into aneural colon, brain‐ and gut‐derived NSCs were able to differentiate into neurons. Conclusions & Inferences Nestin‐expressing intestinal NSCs cells give rise to neurospheres, differentiate into neuronal, glial, and mesenchymal lineages in vitro, generate neurons in vivo and can be isolated from mucosa. Further studies are needed for exploring their potential for treating neuropathies.