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Enteric Glial Cells

Review of Enteric Glial Cell Research


What are enteric glia?   Glial cells are present in our guts.  In fact, our guts possess quite an extensive nervous system.  In the gut, neurons and glia form a complex latticework of cells in the various layers of the intestinal walls, connecting the nerve networks in the muscle and the submucosal layers.  The enteric glia ensheath the cell bodies and axons of enteric neurons.  The enteric glia stain with markers such as S-100 which is also present in central nervous system glial (Kobayashi, et al. 1986).

Enteric nervous system.   Neurons of the gut are organized into groups called myenteric ganglia.  The neurons in these ganglia vary enormously in size and are situated in the muscle layer of the stomach, small intestines, and large intestines.  These ganglia are largest and most complex in the stomach and upper small intestines, smallest in the lower intestines, and medium sized in the large intestines.  Enteric glia are present in these ganglia as well as the network of neural fibers that connect these ganglia (Baluk, et al. 1983; Jessen, et al. 1983).  These cells are abundantly available from the appendix, a potential source of enteric glia for human autografts (Xiong, et al. 2000).

Development of enteric glia.  During development, multipotent cells migrate from the neural crest into the gut (Pomeranz, et al., 1993; Natarajan, et al. 1999).  They possess receptors to neurotrophins, particularly NT-3 which promotes growth and proliferation of the cells (Gershon, 1993; Saffrey, et al. 2000), as well as differentiation into neurons (Chalazonitis, et al, 1994).  This may account for the stimulatory effect of systemically applied neurotrophins on the enteric nervous system (Shen, et al., 1997).  Enteric glia also require GDNF to survive and proliferate (Saarma & Sariola, 1999).  Enteric glial cells produce substantial amounts of cyclic AMP (Christofi, et al., 1993).  In the gut, they have a morphology that is similar to CNS fibrous astrocytes.  They are often star-shaped and have irregularly branched processes in the ganglia but have longer processes that run parallel to neural fibers (Hanani, et al. 1994).  

Culturing enteric glia.   Enteric glia can be readily cultured from adult mammals (Bannerman, et al. 1988; Hanani, 1993; Jaeger, 1995) and can be stimulated to grow and divide in culture for many months (Browsard, et al., 1993).  These cells differ from Schwann cells in several important respects (Gershon & Rothman, 1991).  First, they have different protein markers.  Second, unlike Schwann cells that myelinate individual axons, enteric glia ensheath groups of axons, similar to the way olfactory ensheathing glia ensheath axons in the olfactory bulb (Pixley, 1992).  Enteric glia stimulate neurite outgrowth when they are co-cultured with dissociated neurons (Hopker, et al. 1994).  Enteric glia also respond to a variety of neurotransmitters with increase intracellular calcium entry (Kimball, et al. 1996; Zhang, et al. 1997).

Transplanting enteric glia.  When transplanted into the brain, enteric glia from adults survive and, unlike Schwann cells, do not produce reactive responses in central nervous system astrocytes (Lawrence, et al. 1991).  Jaeger, et al. (1993) were the first to transplant enteric glia into the spinal cord of rats.  Allografts of these cells (from another individual rat), when injected into a rat lower thoracic spinal cord, survive for many weeks and are surrounded by reactive astrocytes and macrophages, possibly a sign of immune response.  Newly formed vasculature penetrate into the grafts.  Tew, et al. (1994) transplanted myenteric plexus ganglia into the corpus striatum (basal ganglia) of rat brains and found that they integrated well into the nervous tissues and allow axonal growth into the grafts. White & Anderson (1999) transplanted neural crest cells and found they generated neurons and glia.  

Recent Studies .  The Society for Neuroscience in San Diego had 11 abstracts of enteric glial cell studies.  The following are several studies from Rathbone's laboratory at McMaster University:  
•  Westiuk, et al. in Canada reported that purines (guanosine, inosine, and dibutyrl cAMP) and neutrophic factors (NT-3, NGF) strongly stimulate proliferation of enteric glia and also enhanced their survival in culture, especially NT-3.  Inosine and NT-3 also stimulate maturation of the cells as indicated by increased expression of the astrocyte marker GFAP within 24 hours.  
•  Wang, et al. transplanted enteric glia to T12 spinal cord after cutting the T12 dorsal roots.  They showed that primary sensory axons entered the spinal cord by 3 weeks after injury and transplantation, invading deep into the spinal cord.  Olfactory ensheathing glial cells had previously been shown to have this effect in spinal cord.
•  Khan, et al. transplanted eneric glia into the spinal cord and examined the migration of the cells in spinal cords at 1-90 days after transplantation.  The cells were labelled with phaseolus vulgaris.  The enteric glia began migrating from the implantation into white mater at 0.6 mm/day up to a month, slowing to 0.4-0.5 mm/day from 2-3 months.  The glia became astrocyte-like and remained in close contat with axons or neurons.  
•  Middlemiss, et al. described methods of culturing enteric glia.  They used a mitotically arrested 3T3 fibroblast feeder layer but had trouble separating the two cell types, so they used inserts (NUNC 0.2) that allowed the two cell types be easily separated.  In culture, the cells fell into two morphological categories:  Type I - flat bipolar/tripolar with ovoid nucleus and short cytoplasmic processes or Type II - irregular cells with round nuclei and long branching cytoplasmic processes.  These cells grew for 50 passages over 12 months in enriched media.
•  Rathbone, et al. further described the morphology of the various enteric glia, separating two populations of glia by their GFAP expression.  Lysophosphatidic acid (10 µM) or bovine serum albumin (10 mg/ml) reversed the ratio of GFAP/vimentim expression and the two types of cells.  The authors suggest that these two cells convert interchangeably.  These morphological categories are similar to the "fried egg", bipolar, and "multipolar" morphological categories of olfactory ensheathing glia.

Summary

1.  Enteric glial cells are present in our guts
2.  They are a component of a complex enteric neural system
3.  Responsive to neurotrophins, these cells look like neurons
4.  They are readily cultured and stimulate neuronal growth in vitro
5.  They continue to proliferate in culture for 12 months or longer
6.  These cells survive transplantation and migrate in spinal cord
7.  Transplanted enteric glia promote regeneration of dorsal roots

Links

http://www.csro.com/3-3.html .  This is a link to the CSRO site that has a video from Michael Rathbone talking about enteric glia.  The video is very short and does not say all that much but it does show a picture of Dr. Rathbone and what he sounds like.

http://www.hosppract.com/issues/1999/07/gershon.htm .  This is an article from Michael Gershon from Columbia University, one of the original pioneers of enteric glial research.  Entitled "The Enteric Nervous System:  A Second Brain", this article summarizes the complex systems that govern our bowel movements.

Reference cited

•  Baluk P, Jessen KR, Saffrey MJ and Burnstock G (1983). The enteric nervous system in tissue culture. II. Ultrastructural studies of cell types and their relationships. Brain Res. 262 (1): 37-47. Summary: 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. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=6831230>

•  Bannerman PG, Mirsky R and Jessen KR (1988). Establishment and properties of separate cultures of enteric neurons and enteric glia. Brain Res. 440 (1): 99-108. Summary: In this paper methods are described for the preparation of two types of culture derived from myenteric explants: (a) highly enriched neuronal cell cultures, and (b) purified glial cells (greater than 98%). Both procedures combine the technique of antibody complement-mediated cytolysis with the use of an antimitotic agent. Immunohistochemical methods were used to compare the purified cells to their counterparts in mixed cultures (see accompanying paper). Antibodies to the glycoprotein Thy-1 and the monoclonal antibody A2B5 which recognizes gangliosides, labelled the cell surface of all enteric neurons in enriched cultures while subpopulations of the neurons expressed the Leu 7 carbohydrate epitope, the neurotransmitter 5-hydroxytryptamine and the neuropeptides substance P, methionine-enkephalin and vasoactive intestinal polypeptide. Autoradiographic experiments show that a subpopulation of enriched neurons exhibit high-affinity uptake sites for gamma-[3H]aminobutyric acid (GABA). All purified enteric glia continue to express the calcium binding protein S100, the basement membrane glycoprotein laminin and the antigens recognized by the A2B5 antibody, and subpopulations of glia are labelled by the monoclonal antibodies LB1 which binds to GD3 gangliosides, and Leu 7. Thus enteric neurons and glia can survive independently of each other and express molecular properties which are present in cultures normally containing both cell types. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2896046> Department of Anatomy and Embryology, University College, London, U.K.

•  Broussard DL, Bannerman PG, Tang CM, Hardy M and Pleasure D (1993). Electrophysiologic and molecular properties of cultured enteric glia. J Neurosci Res. 34 (1): 24-31. Summary: Enteric glia, the support cells of myenteric ganglia, have been widely studied with respect to their morphology and immunohistochemical phenotype, but little is known about their functional properties. We developed a method for the amplification of enteric glia from newborn guinea pigs to further characterize these cells. Treatment with a combination of basic fibroblast growth factor and the adenylate cyclase activator, cholera toxin, permitted expansion of enteric glial cultures to confluence and serial passage for up to 8 months. The long-term cultured cells retained expression of 1) S100 protein, 2) GD3 ganglioside recognized by the monoclonal antibody LB1, and 3) the gene encoding glutamine synthetase. The electrophysiologic properties of cultured enteric glia were studied under whole-cell patch clamp conditions. Most cells expressed "delayed rectifier"-type potassium currents, and some also demonstrated tetrodotoxin-sensitive sodium currents. Other subsets of voltage-dependent potassium currents, calcium currents, and glutamate-gated currents were not demonstrable. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8423634> Division of Gastroenterology, Children's Hospital of Philadelphia, PA 19104.

•  Chalazonitis A, Rothman TP, Chen J, Lamballe F, Barbacid M and Gershon MD (1994). Neurotrophin-3 induces neural crest-derived cells from fetal rat gut to develop in vitro as neurons or glia. J Neurosci. 14 (11 Pt 1): 6571-84. Summary: The precursor cells that form the enteric nervous system (ENS) are multipotent when they arrive in the gut from the neural crest. Their differentiation thus depends on signals from the enteric microenvironment. Crest-derived cells were isolated from the fetal rat bowel by immunoselection at E14 with NC-1/HNK-1 antibodies and secondary antibodies coupled to magnetic beads. NC-1/HNK-1-immunoreactive cells were enriched approximately 36-fold. The NC-1/HNK-1-selected population and the residual population were plated at equal cell density and maintained in a defined medium for 6-7 d. The total number of cells found in the cultures of the residual cells was three- to fourfold that in cultures of immunoselected cells. Neurotrophin-3 (NT-3), but not nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-4/5 (NT-4/5), was found to increase the proportion of neurons (neurofilament-immunoreactive or neuron-specific enolase-immunoreactive) or glia (S-100-immunoreactive) (from 6.6 +/- 0.9% to 15.2 +/- 1.4%; p < 0.001). This effect was concentration dependent (from 1 to 40 ng/ml) and observed only in the cultures of immunoselected cells. NT-3 also enhanced neurite outgrowth. NT-3 increased neither cell number nor bromodeoxyuridine incorporation and thus was not mitogenic. Exposure of immunoselected cells to NT-3 rapidly and transiently induced the appearance of nuclear Fos immunoreactivity. Transcripts coding for TrkC, the transducing receptor for NT-3, were identified in the fetal rat gut (E14-E16) and in the immunoselected population of cells using reverse transcriptase and the polymerase chain reaction. It is concluded that NT-3 specifically promotes the differentiation of enteric crest-derived cells as neurons or glia and may thus play a role in the development and/or maintenance of the ENS. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7965061> Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.

•  Christofi FL, Hanani M, Maudlej N and Wood JD (1993). Enteric glial cells are major contributors to formation of cyclic AMP in myenteric plexus cultures from adult guinea-pig small intestine. Neurosci Lett. 159 (1-2): 107-10. Summary: Cultures derived from ganglia isolated from the small intestine of adult guinea-pigs were used to determine relative contribution of neurons and glial cells to stimulation of cAMP formation by forskolin in myenteric ganglia. In untreated cultures (8-12 days), the ratio of glial cells to neurons was 5-fold higher than the ratio in intact myenteric plexus preparations. Treatment with cytosine arabinoside virtually eliminated the glia by the 12th day. Microelectrode recording of excitatory responses to forskolin in AH/Type 2 neurons confirmed the viability of cultured neurons in cytosine arabinoside. Forskolin elevated the cAMP content of cultures and cytosine arabinoside reduced this effect by 80-90%. This suggests that enteric glial cells are the major contributors to cAMP formation in the cultures and that glial cells contribute significantly to elevation of cAMP levels seen in intact myenteric ganglia. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8264948> Department of Physiology, Ohio State University, College of Medicine, Columbus 43210-1218.

•  Gabella G and Trigg P (1984). Size of neurons and glial cells in the enteric ganglia of mice, guinea-pigs, rabbits and sheep. J Neurocytol. 13 (1): 49-71. Summary: A quantitative light microscopic study has been carried out on the myenteric and submucosal ganglia of the stomach, duodenum, ileum, proximal colon and rectum of the guinea-pig; the enteric ganglia of the ileum were studied also in the mouse, rabbit and sheep. The area of the profiles of nerve cells, of nerve cell nuclei and of glial nuclei, and the proportion of the area of ganglia occupied by neuropil were measured, and the relative numbers of neurons and glial cells were estimated. The myenteric ganglia were found to be firmly anchored to the stroma of the muscle coat; their shape and the shape of their component cells varied with contraction and distension of the musculature. The range of neuronal sizes in the myenteric ganglia was extremely wide. In the guinea-pig, the myenteric neurons were on average largest in the stomach and duodenum and smallest in the ileum, with intermediate values in the colon and rectum; the submucosal neurons showed little variation in average size along the length of the gut. The average size of ganglion neurons in the ileum was greatest in the sheep and smallest in the mouse, and had intermediate values in the guinea-pig and rabbit. The percentage volume of neuropil in the myenteric ganglia was 51% in the mouse, 65% in the guinea-pig, 70% in the rabbit, and 74% in the sheep. The number of glial cells relative to the number of neurons was also ranked in the same order. In all the species examined the submucosal ganglia, when compared with the corresponding myenteric ganglia, had a smaller percentage volume of neuropil, a much smaller number of glial cells and (except in the mouse ileum) neurons of smaller average size. In all the ganglia there was a positive correlation between size of neurons and size of glial cells. The results are discussed in the light of possible relations between body size (and length of the intestine), numerical density of ganglion neurons, average size of neurons, amount of musculature, average distance between neurons, and amount of neuropil. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=6707713>

•  Gershon MD and Rothman TP (1991). Enteric glia. Glia. 4 (2): 195-204. Summary: The structure of the enteric nervous system (ENS) is different from that of extraenteric peripheral nerve. Collagen is excluded from the enteric plexuses and support for neuronal elements is provided by astrocyte-like enteric glial cells. Enteric glia differ from Schwann cells in that they do not form basal laminae and they ensheath axons, not individually, but in groups. Although enteric glia are rich in the S-100 and glial fibrillary acidic proteins, it has been difficult to find a single chemical marker that distinguishes enteric glia from non-myelinating Schwann cells. Nevertheless, two monoclonal antibodies have been obtained that recognize antigens that are expressed on Schwann cells (Ran-1 in rats and SMP in avians) but not enteric glia. Functional differences between enteric glia and non-myelinating Schwann cells, including responses to gliotoxins and in vitro proliferative rates, have also been observed. Developmentally, enteric glia, like Schwann cells, are derived from the neural crest. In both mammals and birds the precursors of the ENS appear to migrate to the bowel from sacral as well as vagal levels of the crest. These crest-derived emigres give rise to both enteric glia and neurons; however, analyses of the ontogeny of the enteric innervation in a mutant mouse (the ls/ls), in which the original colonizing waves of crest-derived precursor cells are unable to invade the terminal colon, suggest that enteric glia can also arise from Schwann cells that enter the gut with the extrinsic innervation. When induced to leave back-transplanted segments of avian bowel, enteric crest-derived cells migrate into peripheral nerves and form Schwann cells. Enteric glia and Schwann cells thus appear to be different cell types, but ones that derive from lineages that diverge relatively late in ontogeny. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1827778> Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032.

•  Hanani M (1993). Neurons and glial cells of the enteric nervous system: studies in tissue culture. J Basic Clin Physiol Pharmacol. 4 (3): 157-79. Summary: The enteric nervous system (ENS) has been recognized as the main component in regulating the function of the digestive tract and as a model for studying neuronal physiology and pharmacology. Most of the present knowledge on the ENS was derived from in vitro studies on freshly isolated plexuses. In 1978 the first study on cultured myenteric neurons was published and since then there has been a growing interest in this method. Several different culture preparations have been introduced, including the recent development of cultures from adult guinea-pigs and humans. This review summarizes the findings which have been made using cultured enteric neurons and glia. The main topics that are described are the role of the extracellular matrix and of hormones on neuronal growth, neuron-glia interactions, release of neuropeptides and their actions on neurons and co-transmission between neurons. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8679514> Laboratory of Experimental Surgery, Hadassah University Hospital, Mount Scopus, Jerusalem, Israel.

•  Hanani M and Reichenbach A (1994). Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig. Cell Tissue Res. 278 (1): 153-60. Summary: Glial cells of the myenteric plexus from guinea pig small intestine were intracellularly filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid somata (8.5 +/- 0.7 microns equivalent diameter, i.e., about 330 micron3 volume), and send up to 20 thin and short processes (less than 26 to about 110 microns in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of "holes" in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of approximately 2000 micron2, almost 90% of which are contributed by the cell processes.(ABSTRACT TRUNCATED AT 250 WORDS). <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7954696> Hadassah University Hospital, Mount Scopus, Jerusalem, Israel.

•  Hopker VH, Saffrey MJ and Burnstock G (1994). Myenteric plexus explants promote neurite elongation and survival of striatal neurons in vitro. Brain Res. 648 (2): 332-6. Summary: Dissociated striatal neurons exhibited increased neurite outgrowth when co-cultured with myenteric plexus explants. Enriched enteric neurons or enriched enteric glia produced a less marked response; non-ganglionic cells had no effect. Increases in striatal neuron and glial cell numbers were seen in all co-cultures. Tetrodotoxin abolished the neuritogenic response of myenteric plexus explants but did not affect increases in cell numbers. These observations suggest that spontaneous neuronal activity within the myenteric plexus is involved in the release of a neuritogenic factor(s), possibly from glial cells, and that this is distinct from the factor(s) affecting striatal cell numbers. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7922550> Department of Anatomy and Developmental Biology, University College London, UK.

•  Jaeger CB, Toombs JP and Borgens RB (1993). Grafting in acute spinal cord injury: morphological and immunological aspects of transplanted adult rat enteric ganglia. Neuroscience. 52 (2): 333-46. Summary: We have studied allogeneic transplants of adult rat enteric ganglia in order to evaluate their use as donor tissue for eventual autografts in rodent spinal cord injury models. Female Sprague-Dawley rats of similar weights served either as transplant donors or as recipients. A glass micropipette of 0.8 mm diameter was used to create a local penetrating injury of the lower thoracic spinal cord and the transplant material was pressure injected through the pipette within the neural parenchyma. Ganglia of the myenteric plexus adhering to the stratum longitudinal muscularis were dissected from portions of the jejunum and ileum. Following partial enzymatic digestion and mechanical disruption of the myenteric plexus and muscle tissue (labeled with adherent rhodamine conjugated microbeads), reaggregates of myenteric plexus and muscle were suspended in growth medium and cultured in vitro for one to two days prior to transplantation. Transplants were examined at three, four, six, and eight weeks after surgery. Some of the donor tissue was grown in vitro, in order to determine its cellular composition. These cultured explants were fixed after 10 days, and like myenteric plexus and muscle grafts, were stained histochemically for acetylcholinesterase and observed by fluorescence and light microscopy. At the earlier post-transplantation periods, grafts contained several clusters of enteric ganglion cells that were positive for acetylcholinesterase and exhibited ultrastructural features characteristic of the enteric nervous system. They had well-defined boundaries. Reactive astrocytes and their processes remained located within the host spinal cord adjacent to the boundary region of the grafts. Likewise, macrophages were located in areas abutting the graft. Newly formed vasculature penetrated the graft interior and appeared to be continuous with the host vessels. Grafts grown for at least eight weeks were characterized by interdigitating boundaries. Finger-like protrusions of graft tissue containing fibroblasts and collagen intermixed with adjacent gray and white matter of the host cord. Such transplants also had reactive astrocytes and ED1-positive macrophages. At this later stage, several groups of ganglion cells were identified that were intensely acetylcholinesterase-positive; however, only two of four grafts were recovered, whereas two of the transplants degenerated. We postulate that degeneration of allogeneic grafts may occur as a result of ongoing immune responses of the host which could be prevented by use of autogeneic enteric ganglia. Our studies show that fully differentiated enteric ganglia can survive transplantation to acutely injured spinal cord of adult rats. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8450950> Department of Anatomy, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

•  Jessen KR, Saffrey MJ and Burnstock G (1983). 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. Brain Res. 262 (1): 17-35. Summary: 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. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=6831228>

•  Kimball BC and Mulholland MW (1996). Enteric glia exhibit P2U receptors that increase cytosolic calcium by a phospholipase C-dependent mechanism. J Neurochem. 66 (2): 604-12. Summary: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 mM), glutamate (100 microM), norepinephrine (10 microM), and substance P (1 microM) did not increase the intracellular calcium concentration ([Ca2+]i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 microM), bradykinin (11%; 10 microM), and histamine (31%; 100 microM), whereas 100% of glia responded to ATP (100 microM). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca2+]i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca2+]i evoked, ATP = UTP > ADP > beta, gamma-methyleneadenosine 5'-triphosphate >> 2-methylthioadenosine 5'-triphosphate = alpha,beta-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P2U receptor. Depletion of D-myo-inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 microM) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 microM), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca2+]i. Our data suggest that glial responses to ATP are mediated by a P2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8592130> Department of Surgery, University of Michigan, Ann Arbor, USA.

•  Kobayashi S, Suzuki M, Endo T, Tsuji S and Daniel EE (1986). Framework of the enteric nerve plexuses: an immunocytochemical study in the guinea pig jejunum using an antiserum to S-100 protein. Arch Histol Jpn. 49 (2): 159-88. Summary: Immunostained sections and whole-mount preparations of the layers of the guinea pig jejunum were investigated by an improved peroxidase-antiperoxidase method using an antiserum to S-100 protein. A delicate latticework of S-100 protein immunopositive glial cells was demonstrated extending in the longitudinal muscle layer, myenteric or Auerbach's plexus, circular muscle layer including the deep muscular plexus, submucous layer including the submucous or Meissner's plexus, lamina muscularis mucosae and lamina propria mucosae. The whole enteric nerve plexuses consisted of two subsystems; nerve plexuses of the muscular coat and those of the submucous and mucous coats. These two subsystems were joined to each other by thick, connecting branches perforating the inner circular muscle layer. Extrinsic nerves entering the myenteric plexus formed a specialized junctional structure containing S-100 protein immunopositive glial cells, whereas those entering the submucous plexus ran along the submucous arteries. We proposed the term enteroglial cells to designate the S-100 protein immunopositive cells which ensheathed the somata and processes of the enteric neurons. The frameworks of all structures in the enteric nerve plexuses from the largest ganglia to the thinnest nerve fasciculi were constructed of these enteroglial cells. A spectrum of the enteroglial cells was presented. Those in the myenteric and submucous ganglia were found similar to the astroglia of the central nervous system and to the satellite cells in the peripheral ganglia. Those in the primary and secondary fasciculi of the myenteric plexus formed a kind of neuropil together with the neuronal processes. Those in the tertiary fasciculi of the muscular coat formed the framework of the autonomic ground plexus. We tentatively concluded that the interstitial cells of Cajal contain an immunoreactivity for S-100 protein, and thus are glial in nature. The occurrence of specialized enteroglial cells with a neuron-like function was discussed in the autonomic ground plexus of the muscular coat. In the lamina propria mucosae, there was a fine latticework of the S-100 protein immunopositive enteroglial cells. This latticework corresponded to that of the interstitial cells of Cajal in the villous and periglandular plexuses. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=3532998>

•  Lawrence JM, Raisman G, Mirsky R and Jessen KR (1991). Transplantation of postnatal rat enteric ganglia into denervated adult rat hippocampus. Neuroscience. 44 (2): 371-9. Summary: These experiments explore the possible value of the myenteric plexus as a source of donor cells for autografting into the central nervous system. Neurons and glia from 10-12-day postnatal rat myenteric plexus survive for at least one month after transplantation into cholinergically denervated syngeneic adult rat hippocampus. A population of donor cholinergic neurons has acetylcholinesterase-positive processes, but these appear not to innervate host tissue. Host gliosis in response to these implants seems to be less than that seen with other peripheral ganglia, and unlike Schwann cells, the enteric glia form end-feet on brain capillaries. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1944891> Norman and Sadie Lee Research Centre, Laboratory of Neurobiology, National Institute for Medical Research, Mill Hill, London, U.K.

•  Natarajan D, Grigoriou M, Marcos-Gutierrez CV, Atkins C and Pachnis V (1999). Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture. Development. 126 (1): 157-68. Summary: The enteric nervous system of vertebrates is derived from neural crest cells that invade the gut wall and generate a highly organised network of enteric ganglia. Among the genes that play an important role in ENS development is c-Ret, mutations of which result in failure of formation of enteric ganglia (intestinal aganglionosis). To further understand the development of the mammalian ENS in general and the mechanism of action of the RET RTK in particular, we have developed and used an organotypic culture system of mouse fetal gut. At the stage of culture initiation, the gut is partially populated by undifferentiated ENS progenitors, but culture for several days results in extensive neuronal and glial differentiation. Using this organ culture system, we have compared the development of the ENS in wild-type and RET-deficient gut and showed that the aganglionic phenotype observed in vivo is consistently reproduced under the in vitro culture conditions. Microinjection of RET+ cells isolated from E11.5 mouse bowel into wild-type or RET-deficient aganglionic gut in organ culture, results in extensive repopulation of their wall by exogenously derived neurons and glia. Finally, using a similar approach, we demonstrate that single RET+ cells introduced into the wall of wild-type gut generate both cell lineages of the ENS, i.e. neurons and glia. Our data show the NC-derived RET+ population of fetal gut in mammalian embryos consists of multipotential progenitors capable of colonising efficiently both wild-type and RET-deficient aganglionic bowel in organ culture. <http://www.biologists.com/Development/126/01/dev1344.html
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9834195> Divisions of Developmental Neurobiology and Cellular Immunology, MRC, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK.

•  Pixley SK (1992). The olfactory nerve contains two populations of glia, identified both in vivo and in vitro. Glia. 5 (4): 269-84. Summary: The peripheral olfactory nervous system exhibits, uniquely, neuronal cell body replacement and reestablishment of central connections in adult mammals. The role of the olfactory nerve glia in these phenomena is unknown, but information might be provided by in vitro systems. This paper reports on the characterization of olfactory nerve glia in dissociated cell cultures of newborn rat nasal mucosal tissues. The predominant type of glial cell resembled Schwann cells and immunostained for the S-100 protein, found in all glial cell types; glial fibrillary acidic protein (GFAP), found in astrocytes and nonmyelinating Schwann cells; and showed binding of 217C, a monoclonal Schwann-cell marker that binds to the low-affinity NGF receptor in glioma cells. They were negative for A2B5. The Schwann-cell-like olfactory glia changed morphology upon culturing in serum-free medium, with further shape changes after plating on laminin. Plating on laminin increased cell numbers. A second population, found only after GFAP-immunostaining, was astrocyte-like in morphology and represented approximately 10 percent of all glial cells. These were S-100-, A2B5-, and 217C-negative, a unique glial cell immunological profile. At low dilutions of anti-GFAP (1/10,000), or with weak fluorescent secondary antibodies, astrocyte-like glia were immunostained but Schwann-cell-like glia were not detectable. Astrocyte-like glia were not an artifact of the dissection, since they were detectable in tissue sections of newborn-rat olfactory nerves immunostained with a low dilution of anti-GFAP. The presence of two types of glial cells in culture suggests similarities between olfactory glia and enteric glia. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1534071> Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, Ohio 45267-0521.

•  Jaeger CB (1995). Isolation of enteric ganglia from the myenteric plexus of adult rats. J Neural Transplant Plast. 5 (4): 223-32. Summary: Enteric neurons and glia cells were isolated from adult Sprague Dawley rats. A procedure is described using a combination of microdissection and mechanical dissociation after enzyme treatment which yields large numbers of cell clusters suitable for tissue culture and grafting into the injured spinal cord. Differentiated enteric ganglia remained viable for at least 5 days in vitro. Cultured neurons expressed histochemical reactivity for acetylcholinesterase and nicotinamide adenine dinucleotide phosphate diaphorase. Nestin positive glia, which represented a population of non-myelinating enteric Schwann cells, could also be identified in cultures maintained 5 days or longer in vitro. The myenteric plexus of adult rats can provide a readily available source of neurons and Schwann cells for grafting to the central nervous system. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7578438> Center for Paralysis Research, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA.

•  Pomeranz HD, Rothman TP, Chalazonitis A, Tennyson VM and Gershon MD (1993). Neural crest-derived cells isolated from the gut by immunoselection develop neuronal and glial phenotypes when cultured on laminin. Dev Biol. 156 (2): 341-61. Summary: The neural crest-derived cells that colonize the bowel are different from their predecessors in the premigratory crest. A procedure, which utilized the immunoselection of cells with a magnet, was thus devised to obtain crest-derived precursors from developing gut. Primary antibodies against cell surface antigens, NC-1 in chick, quail, and rat, or antibodies to a 110-kDa laminin binding protein (alpha-110) in mouse, were used in conjunction with secondary antibodies coupled to magnetic beads. Immediately after immunoselection with NC-1, almost all of the selected cells were NC-1-immunoreactive. Neurons and glia, identified immunocytochemically with antibodies to specific markers, developed preferentially in cultures of immunoselected cells. Some of the phenotypes expressed by neurons arising in vitro were appropriate for the bowel (serotonin- and vasoactive intestinal peptide-immunoreactive); however, catecholaminergic neurons, which are not present in the enteric nervous system, also differentiated in the cultures. Neuronal development, as well as neurite outgrowth, were promoted by laminin. Cells selected with alpha-110 from the fetal murine bowel preferentially gave rise in vitro to neurons and glia. These data suggest that the population of crest-derived cells that colonizes the gut is multipotent, that development of catecholaminergic neurons in situ is prevented by the intact enteric microenvironment, that laminin is important in the formation of enteric ganglia, and that the 110-kDa laminin binding protein is expressed on the surfaces of the immediate precursors of enteric neurons and glia. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8462736> Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York, New York 10032.

•  Saarma M and Sariola H (1999). Other neurotrophic factors: glial cell line-derived neurotrophic factor (GDNF). Microsc Res Tech. 45 (4-5): 292-302. Summary: Glial cell line-derived neurotrophic factor (GDNF) was first discovered as a potent survival factor for midbrain dopaminergic neurons and was then shown to rescue these neurons in animal models of Parkinson's disease. GDNF is a more potent survival factor for dopaminergic neurons and the noradrenergic neurons of the locus coeruleus than other neurotrophic factors, and an almost 100 times more efficient survival factor for spinal motor neurons than the neurotrophins. The members of the GDNF family, GDNF, neurturin (NTN), persephin (PSP), and artemin (ART), have seven conserved cysteine residues with similar spacing, making them distant members of the transforming growth factor-beta (TGF-beta) superfamily. Like the members of the neurotrophin family, the GDNF-like growth factors belong structurally to the cysteine knot proteins. Like neurotrophins, GDNF family proteins are responsible for the development and maintenance of various sets of sensory and sympathetic neurons but, in addition, GDNF and NTN are also responsible for the development and survival of the enteric neurons, and NTN for parasympathetic neurons. All neurotrophins bind to the p75 low-affinity receptor, but their ligand specificity is determined by trk receptor tyrosine kinases. GDNF, NTN, PSP, and ART mediate their signals via a common receptor tyrosine kinase, Ret, but their ligand specificity is determined by a novel class of glycosylphosphatidylinositol (GPI)-anchored proteins called the GDNF family receptor alpha (GFR alpha). GDNF binds preferentially to GFR alpha1, NTN GFR alpha2, ART GRF alpha3, and PSP GFR alpha4 as a co-receptor to activate Ret. GFR alpha4 has until now been described only from chicken. Although the GDNF family members signal mainly via Ret receptor tyrosine kinase, there is recent evidence that they can also mediate their signals via GFR alpha receptors independently of Ret. The GDNF family of growth factors, unlike neurotrophins, has a well-defined function outside the nervous system. Recent transgenic and organ culture experiments have clearly demonstrated that GDNF is a mesenchyme-derived signaling molecule for the promotion of ureteric branching in kidney development. NTN, ART, and PSP are also expressed in the developing kidney, and NTN and PSP induce ureteric branching in vitro, but their true in vivo role in kidney morphogenesis is still unclear. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10383122> Program for Molecular Neurobiology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland. Mart.Saarma@helsinki.fi

•  Saffrey MJ, Wardhaugh T, Walker T, Daisley J and Silva AT (2000). Trophic actions of neurotrophin-3 on postnatal rat myenteric neurons in vitro. Neurosci Lett. 278 (3): 133-6. Summary: A number of neurotrophic factors have been implicated in the prenatal development of the enteric nervous system. Although several of these factors continue to be expressed in the gut during postnatal life, their actions on postnatal enteric neurons are not understood. One such factor is the neurotrophin, NT-3. Both NT-3 and its high affinity receptor, trk C, are expressed in the postnatal gut at a time when changes in the density of intestinal innervation are occurring. We have therefore examined the effects of NT-3 on postnatal myenteric neurons, using dissociated cell cultures of ganglia isolated from 6-8 day postnatal rat small intestine. Effects of NT-3 on neurite outgrowth and neuronal and glial cell numbers were measured after 2 days in vitro. The proportion of neurons was increased in NT-3 treated cultures, as was the proportion of neurons that extended processes. NT-3 treatment, at concentrations of between 0.1 ng and 10 ng/ml, also resulted in a significant increase in mean total neurite length. These results indicate that NT-3 may play a role in the postnatal development of the enteric nervous system. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10653011> Department of Biology, The Open University, Milton Keynes, UK. j.saffrey@open.ac.uk

•  Shen L, Figurov A and Lu B (1997). Recent progress in studies of neurotrophic factors and their clinical implications. J Mol Med. 75 (9): 637-44. Summary: Neurotrophic factors are endogenous soluble proteins that regulate long-term survival and differentiation of neurons of the peripheral and central nervous systems. These factors play an important role in the structural integrity of the nervous system, and therefore are good candidates as therapeutic agents for neurodegenerative diseases. Howev
er, recent studies have revealed some unexpected, novel roles of neurotrophic factors. Of particular significance is the discovery of the new functions of brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). Physiological experiments indicate that BDNF may serve as regulatory factors for synaptic transmission as well as for learning and memory. Gene targeting studies demonstrate that GDNF may be essential for development of the enteric nervous system (ENS) and kidney organogenesis. These results not only provide new insights into our understanding of the function of neurotrophic factors but may also have significant implications in the therapeutic usages of neurotrophic factors. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9351702> Laboratory of Mammalian Gene and Development, NICHD, National Institutes of Health, Bethesda, MD 28920-4480, USA.

•  White PM and Anderson DJ (1999). In vivo transplantation of mammalian neural crest cells into chick hosts reveals a new autonomic sublineage restriction. Development. 126 (19): 4351-63. Summary: The study of mammalian neural crest development has been limited by the lack of an accessible system for in vivo transplantation of these cells. We have developed a novel transplantation system to study lineage restriction in the rodent neural crest. Migratory rat neural crest cells (NCCs), transplanted into chicken embryos, can differentiate into sensory, sympathetic, and parasympathetic neurons, as shown by the expression of neuronal subtype-specific and pan-neuronal markers, as well as into Schwann cells and satellite glia. In contrast, an immunopurified population of enteric neural precursors (ENPs) from the fetal gut can also generate neurons in all of these ganglia, but only expresses appropriate neuronal subtype markers in Remak's and associated pelvic parasympathetic ganglia. ENPs also appear restricted in the kinds of glia they can generate in comparison to NCCs. Thus ENPs have parasympathetic and presumably enteric capacities, but not sympathetic or sensory capacities. These results identify a new autonomic lineage restriction in the neural crest, and suggest that this restriction preceeds the choice between neuronal and glial fates. <http://www.biologists.com/Development/126/19/dev3978.html
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10477302> Division of Biology 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.

•  Xiong S, Puri P, Nemeth L, O'Briain DS and Reen DJ (2000). Neuronal hypertrophy in acute appendicitis. Arch Pathol Lab Med. 124 (10): 1429-33. Summary: OBJECTIVE: The pathogenesis of appendicitis remains poorly understood. However, there is increasing evidence of involvement of the enteric nervous system in immune regulation and in inflammatory responses. This study was set up to characterize the status of the enteric nervous system in normal and in inflamed appendixes. METHODS: S100- and 2',2'-cyclic nucleotide 3' phosphodiesterase-positive Schwann cells, synaptophysin, and neuron-specific, enolase-positive nerve fibers and tryptase-positive mast cells were evaluated with immunohistochemical staining in surgically resected appendixes from 20 children with histologically proven acute appendicitis (HA), 10 histologically normal appendixes (HN) from patients with a clinical diagnosis of appendicitis, and 10 normal appendixes from patients undergoing elective abdominal surgery. Immunostained sections were subjected to quantitative image analysis. The number and size of ganglia and the number of nerve fibers, Schwann cells, and mast cells in each tissue compartment was quantitatively or semiquantitatively measured. RESULTS: Increased numbers of fibers, Schwann cells, and enlarged ganglia, widely distributed in the muscularis externa and submucosa, were seen in all HA appendixes and in 4 of 10 HN appendixes. The number and size of ganglia in muscularis externa and in the submucosa of appendixes with HA were significantly greater compared with those in control appendixes (P <.001). A significantly increased number of individually stained nerve fibers and Schwann cells (P <.05) were present in the muscularis externa in HA appendixes compared with control appendixes. Significantly increased numbers of tryptase-positive mast cells (P <.05) were present in the submucosa, muscularis, and especially in the lamina propria in HA specimens, compared with that of control tissue. CONCLUSIONS: The significant increase in neural components and mast cells in acute appendicitis is unlikely to develop during a single acute inflammatory episode. This suggests an underlying chronic abnormality as a secondary reaction to repeated bouts of inflammation, obstruction, or both. These results challenge our current understanding of the pathophysiological processes that give rise to acute appendicitis. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11035570> Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland.

•  Zhang W, Sarosi G, Jr., Barnhart D, Yule DI and Mulholland MW (1997). Endothelin-activated calcium signaling in enteric glia derived from neonatal guinea pig. Am J Physiol. 272 (5 Pt 1): G1175-85. Summary: The ability of guinea pig enteric glia to respond to endothelins was examined using fura 2-based digital microscopy in glial cells derived from guinea pig taenia coli. Each isoform of endothelin (ET-1, ET-2, ET-3) evoked dose-dependent and equipotent increases in intracellular Ca2+ concentration ([Ca2+]i) and in percentage of cells responding, 4alaEt-1, an ETB receptor agonist, elicited similar [Ca2+]i increments. BQ-788, an ETB antagonist, inhibited [Ca2+]i responses to endothelin. Preincubation of glia with U-73122 a phospholipase C inhibitor, abolished the [Ca2+]i response to ET-3 exposure. Thapsigargin also eliminated ET-3-evoked Ca2+ signaling. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, introduced into glial cells by radio frequency electroporation, blocked [Ca2+]i responses to ET-3 (100 nM) in 63% of glia. Sustained elevation in [Ca2+]i was abolished by removal of Ca2+ from the buffer and inhibited 85. -3% by Ni2+ (1 mM). Preincubation of glia with 100 nM phorbol 12-myristate 13-acetate (24 h) also inhibited sustained increments in [Ca2+]i by 87%. The presence of IP3 receptors in enteric glia was confirmed by immunofluorescent confocal microscopy. Powered by vBadvanced CMPS v4.2.1