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     

Structurally defined signaling in neuro-glia units in the enteric nervous system

Coordination of gastrointestinal function relies on joint efforts of enteric neurons and glia, whose crosstalk is vital for the integration of their activity. To investigate the signaling mechanisms and to delineate the spatial aspects of enteric neuron-to-glia communication within enteric ganglia we developed a method to stimulate single enteric neurons while monitoring the activity of neighboring enteric glial cells. We combined cytosolic calcium uncaging of individual enteric neurons with calcium imaging of enteric glial cells expressing a genetically encoded calcium indicator and demonstrate that enteric neurons signal to enteric glial cells through pannexins using paracrine purinergic pathways. Sparse labeling of enteric neurons and high-resolution analysis of the structural relation between neuronal cell bodies, varicose release sites and enteric glia uncovered that this form of neuron-to-glia communication is contained between the cell body of an enteric neuron and its surrounding enteric glial cells. Our results reveal the spatial and functional foundation of neuro-glia units as an operational cellular assembly in the enteric nervous system.     

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.     

Human mast cell mediator cocktail excites neurons in human and guinea-pig enteric nervous system

Neuroimmune interactions are an integral part of gut physiology and involved in the pathogenesis of inflammatory and functional bowel disorders. Mast cells and their mediators are important conveyors in the communication from the innate enteric immune system to the enteric nervous system (ENS). However, it is not known whether a mediator cocktail released from activated human mast cells affects neural activity in the ENS. We used the Multi-Site Optical Recording Technique to image single cell activity in guinea-pig and human ENS after application of a mast cell mediator cocktail (MCMC) that was released from isolated human intestinal mucosa mast cells stimulated by IgE-receptor cross-linking. Local application of MCMC onto individual ganglia evoked an excitatory response consisting of action potential discharge. This excitatory response occurred in 31%, 38% or 11% neurons of guinea-pig submucous plexus, human submucous plexus, or guinea-pig myenteric plexus, respectively. Compound action potentials from nerve fibres or fast excitatory synaptic inputs were not affected by MCMC. This study demonstrates immunoneural signalling in the human gut and revealed for the first time that an MCMC released from stimulated human intestinal mast cells induces excitatory actions in the human and guinea-pig ENS.     

Relationship between enteric neurons and interstitial cells in the primate gastrointestinal tract

Background Morphological studies have revealed a close anatomical relationship between enteric nerve terminals and intramuscular ICC (ICC‐IM) which supports a role for ICC‐IM as intermediaries in enteric motor neurotransmission. Recently, a second type of interstitial cell previously described as ‘fibroblast‐like’ but can now be identified by platelet‐derived growth factor receptor‐α expression, has also been implicated in enteric neurotransmission in rodents. The present study was performed to determine if enteric nerve fibers form close anatomical relationships with ICC and PDGFRα⁺ cells throughout the primate GI tract. Methods Immunohistochemical experiments and confocal microscopy were performed to examine the relationship between excitatory and inhibitory motor neurons, ICC and PDGFRα⁺ cells throughout the monkey GI tract. Key Results The pan neuronal marker. Protein gene product 9.5 (PGP9.5) was used to label all enteric neurons and substance‐P (sub‐P) and neuronal nitric oxide synthase (nNOS) to label excitatory and inhibitory neurons, respectively. Double labeling with Kit revealed that both classes of nerve fibers were closely apposed with ICC‐IM in the stomach, small intestine and colon (taenia and inter‐taenia regions), but not with ICC at the level of the myenteric plexus (ICC‐MY). Varicose enteric nerve fibers were closely associated with ICC‐IM for distances up to 250 μm. Both excitatory and inhibitory nerve fibers were also closely apposed to PDGFRα⁺ cells throughout the primate GI tract. Conclusions & Inferences The close anatomical relationship between enteric nerve fibers and ICC‐IM and PDGFRα⁺ cells throughout the GI tract of the Cynomolgus monkey provides morphological evidence that these two classes of interstitial cells may provide a similar physiological function in primates as has been attributed in rodent animal models.     

Expression of a functional metabotropic glutamate receptor 5 on enteric glia is altered in states of inflammation

The metabotropic glutamate receptor 5 (mGluR5) is expressed by astrocytes and its expression is modulated by inflammation. Enteric glia have many similarities to astrocytes and are the most numerous cell in the enteric nervous system (ENS). We investigated whether enteric glia express a functional mGluR5 and whether expression of this receptor was altered in colitis. In both enteric plexuses of the ileum and colon of guinea pigs and mice, we observed widespread glial mGluR5 expression. Incubation of isolated segments of the guinea pig ileum with the mGluR5 specific agonist RS-2-chloro-5-hydroxyphenylglycine (CHPG) caused a dose-dependent increase in the glial expression of c-Fos and the phosphorylated form of the extracellular signal-regulated kinase 1/2. Preincubation of tissues with the group I metabotropic glutamate receptor antagonist, S-4-carboxyphenylglycine, abolished the effects of CHPG. We examined mGluR5 expression in the guinea pig trinitrobenzene sulfonic acid and the IL-10 gene-deficient (IL-10(-/-)) mouse models of colitis. In guinea pigs, mGluR5 immunoreactivity became diffusely localized over the colonic myenteric ganglia, suggesting a change in receptor distribution. In contrast, glial mGluR5 expression was significantly reduced in the colonic myenteric plexus of IL-10(-/-) mice, as assessed with both real-time quantitative RT-PCR as well as immunohistochemistry and image analysis. These changes occurred without concomitant changes to enteric ganglia or glial fibrillary acidic protein expression in the IL-10(-/-) mouse. Our data suggest that enteric glia are a functional target of the glutamatergic neurotransmitter system in the ENS and that changes in mGluR5 expression may be of physiological significance during colitis.     

The enteric nervous system in tissue culture. II. Ultrastructural studies of cell types and their relationships

Tissue culture preparations of the myenteric plexus from the guinea pig taenia coli have been studied by electron microscopy. Three main cell types can be identified: neurons, enteric glial cells and fibroblasts. The ultrastructure of these cells resembles that of the same cells in situ. Neuronal processes form close associations with other neurons and glial cells, but not with fibroblasts. After extended periods in culture, neurons and glial cells form aggregates of cells which resemble ganglia of the myenteric plexus in situ, having a compact neuropil and synapses between neuronal elements. Aggregates are connected to each other by thick bundles of neurites. Vesicle-containing nerve profiles are common; the majority contain a predominance of small agranular vesicles, but some contain many large granular or large opaque vesicles; profiles may also contain variable mixtures of these kinds of vesicles.     

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.     

Macrophages and glia participate in the removal of apoptotic neurons from the Drosophila embryonic nervous system

Cell death in the Drosophila embryonic central nervous system (CNS) proceeds by apoptosis, which is revealed ultrastructurally by nuclear condensation, shrinkage of cytoplasmic volume, and preservation of intracellular organelles. Apoptotic cells do not accumulate in the CNS but are continuously removed and engulfed by phagocytic haemocytes. To determine whether embryonic glia can function as phagocytes, we studied serial electron microscopic sections of the Drosophila CNS. Apoptotic cells in the nervous system are engulfed by a variety of glia including midline glia interface (or longitudinal tract) glia, and nerve root glia. However, the majority of apoptotic cells in the CNS are engulfed by subperineurial glia in a fashion similar to the microglia of the vertebrate CNS. A close proximity between macrophages and subperineurial glia suggests that glia may transfer apoptotic profiles to the macrophages. Embryos affected by the maternal-effect mutation Bicaudal-D have no macrophages. In the absence of macrophages, most apoptotic cells are retained at the outer surfaces of the CNS, and subperineurial glia contain an abundance of apoptotic cells. Some apoptotic cells are expelled from the CNS, which suggests that the removal of apoptotic cells can occur in the absence of macrophages. The number of subperineurial glia is unaffected by changes in the rate of neuronal apoptosis. © 1995 Wiley-Liss, Inc.     

Neural stem cell transplantation in the enteric nervous system: roadmaps and roadblocks

Abstract  The enteric nervous system (ENS) is vulnerable to a variety of genetic, metabolic or environmental threats, resulting in clinical disorders characterized by loss or malfunction of neuronal elements. These disorders have been difficult to treat and there is much enthusiasm for novel therapies such as neural stem cell (NSC) transplantation to restore ENS function in diseased segments of the gut. Recent research has indicated the potential for a variety of innovative approaches to this effect using NSC obtained from the central nervous system (CNS) as well as gut derived enteric neuronal progenitors. The main goal of this review is to summarize the current status of NSC research as it applies to the ENS, delineate a roadmap for effective therapeutic strategies using NSC transplantation and point out the numerous challenges that lie ahead.     

Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerability

Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+-free medium, NMDA (10 microM; 3-5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 microM) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mM) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition.     

The enteric nervous system in tissue culture. I. Cell types and their interactions in explants of the myenteric and submucous plexuses from guinea pig, rabbit and rat

This paper describes methods for removing the ganglionated myenteric and submucous plexuses from the mammalian gut and maintaining them as explants in tissue culture. A detailed account is given of cell types, their interactions and the development of these cultures during 5 weeks in vitro.Three major cell types were identified in the cultures: neurons, glial cells and fibroblasts. The development of the plexuses in culture was studied in detail for the myenteric plexus from the guinea pig taenia coli. It followed a characteristic pattern, in which the merging of individual ganglia into a continuous monolayer of flattened neurons was accompanied and followed by the formation of an extensive outgrowth zone of flat glial cells covered by a dense mesh of outgrowing neurites. In older cultures, neuronal migration resulted in the reformation of discrete and compact aggregates, which consisted of neurons and glial cells, and were interconnected by thick neurite bundles. This arrangement resembles in many ways the original organization of enteric nervous tissue in vivo.This is the first time the enteric ganglia have been freed from the gut wall and grown in culture as explants of nervous tissue. These preparations open many new directions for investigations of the largest and most complex division of the peripheral nervous system, including studies of the molecular nature of neuronal and glial cell surfaces,analysis of cell-cell interactions, trophic factors and developmental signals.     

Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones

Abstract Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB₁ receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI‐related drug development such as in obesity treatment. The aim of the study was to investigate how CB₁ ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea‐pig myenteric neurones were loaded with different fluorescent markers: Fluo‐4 to measure network activity, FM1‐43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB₁ receptors was confirmed by RT‐PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB₁ receptor antagonists (AM‐251 and SR141716), abolished by CB₁ activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA‐5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB₁ agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin‐positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB₁ receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.     

Chronic interleukin-6 alters the level of synaptic proteins in hippocampus in culture and in vivo

There is now considerable evidence that the level of expression of the proinflammatory cytokine, interleukin-6 (IL-6), is increased in the central nervous system (CNS) during neuroinflammatory conditions such as occurs in neurological disorders and in disease and injury. However, our understanding of the consequences of increased expression of IL-6 on the CNS is still limited, especially with respect to the developing nervous system, which is known to be particularly vulnerable to environmental factors. To address this issue, we investigated the properties of cultured hippocampal neurons exposed chronically to IL-6 during the main period of morphological and physiological development, which occurs during the first 2 weeks of culture. IL-6 was tested at 500 U/mL, considered to reflect a pathophysiologic concentration. The morphological features of neuronal development in the control and IL-6-treated cultures appeared similar. However, Western blot analysis showed a significant reduction in the level of Group-II metabotropic receptors (mGluR2/3) and L-type Ca(2+) channels in the IL-6-treated cultures. A similar reduction in mGluR2/3 and L-type Ca(2+) channel protein was observed in transgenic mice that over-express IL-6 in the CNS through astrocyte production starting early in development. Analysis of Ca(2+) signals produced by spontaneous synaptic network activity in the hippocampal cultures and effects of a mGluR2/3 agonist and antagonist showed that the reduced levels of mGluR2/3 impact on the functional properties of hippocampal synaptic network activity. These results have important implications relative to the mechanisms responsible for altered CNS function during conditions associated with increased levels of IL-6 in the CNS.