Enteric glia.

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 emigrés 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.     

Glial cell lineages in the neural crest.

We have been studying how and when the different peripheral glial cell lineages individualize during avian embryonic development. Three different and complementary experimental approaches were used for this purpose: 1) the quail/chick chimera system allowed the tracing in vivo of the origin of the various types of peripheral glial cells (Schwann cells of nerves, satellite glial cells of sensory and autonomic ganglia, and enteric glial cells), and the analysis of the non-neuronal cell population of ganglia; 2) characterisation of early cell-type specific markers that discriminate between the different glial cell subpopulations; and 3) analysis of the progeny of neural crest cells in clonal cultures. As a result of these approaches, two novel glial-specific markers, expressed earlier than any previously described myelin components, have been identified and partly characterised. The divergence of glial and neuronal cell lineages is a process that is not completely terminated during the phase of neural crest migration. Whereas some cells are apparently already totally committed to a glial fate at this stage, others retain dual neuronal/glial potentialities.     

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.     

Negative Regulation of Schwann Cell Myelin Protein Gene Expression by the Dorsal Root Ganglionic Microenvironment

In vivo , the surface glycoprotein Schwann cell myelin protein (SMP) is expressed in the quail peripheral nervous system exclusively by Schwann cells. It is not detectable at any developmental stage either in enteric glia or in ganglionic satellite cells. We demonstrate here that the satellite glial cells of the dorsal root ganglia start to express SMP on their surface when they are dissociated into single cells and cultivated in vitro. Activation of SMP synthesis is a rapid event observed in mass cultures of dorsal root ganglia dissociated cells as soon as 4 h after the onset of the culture. Confocal microscope analysis revealed that satellite cells may acquire the Schwann cell marker when still in close contact with the neuronal soma. Clonal cultures of satellite cells from E8 dorsal root ganglia demonstrated that the progeny of these SMP-negative cells steadily express SMP. This, together with similar results previously obtained with enteric glia, suggests that the SMP-positive phenotype is a constitutive trait of the peripheral glial cell lineage which is inhibited in satellite cells in vivo by the microenvironment prevailing in the peripheral nervous system ganglia.     

Radial glial cells as neuronal precursors: a new perspective on the correlation of morphology and lineage restriction in the developing cerebral cortex of mice

Radial glia is a ubiquitous cell type in the developing central nervous system (CNS) of vertebrates, characterized by radial processes extending through the wall of the neural tube which serve as guiding cables for migrating neurons. Radial glial cells were considered as glial precursor cells due to their astroglial traits and later transformation into astrocytes in the mammalian CNS. Accordingly, a hypothetical morphologically distinct type of precursor was attributed the role of neurogenesis. Recent evidence obtained in vitro and in vivo, however, revealed that a large subset of radial glia generates neurons. We further demonstrate here that the progeny of radial glial cells does not differ from the progeny of precursors labeled from the ventricular surface, implying that there is no obvious relation between precursor morphology and neuron-glia lineage decisions in the developing cerebral cortex of mice. Moreover, we show that many radial glial cells seem to maintain their process during cell division and discuss the implications of this observation for the orientation of cell division. These new data are then related to radial glial cells in other non-mammalian vertebrates persisting into adulthood and suggest that radial glia are not only neurogenic during development, but also in adulthood.     

Lysophosphatidic acid activates satellite glia cells and Schwann cells

Pruritus is a common and disabling symptom in patients with hepatobiliary disorders, particularly in those with cholestatic features. Serum levels of lysophosphatidic acid (LPA) and its forming enzyme autotaxin were increased in patients suffering from hepatic pruritus, correlated with itch severity and response to treatment. Here we show that in a culture of dorsal root ganglia LPA 18:1 surprisingly activated a large fraction of satellite glia cells, and responses to LPA 18:1 correlated inversely with responses to neuronal expressed transient receptor potential channels. LPA 18:1 caused only a marginal activation of heterologously expressed TRPV1, and responses in dorsal root ganglion cultures from TRPV1-deficient mice were similar to controls. LPA 18:1 desensitized subsequent responsiveness to chloroquine and TGR5 agonist INT-777. The LPA 18:1-induced increase in cytoplasmatic calcium stems from the endoplasmatic reticulum. LPA receptor expression in dorsal root ganglia and Schwann cells, LPAR1 immunohistochemistry, and pharmacological results indicate a signaling pathway through LPA receptor 1. Peripheral rat Schwann cells, which are of glial lineage as the satellite glia cells, were also responsive to LPA 18:1. Summarizing, LPA 18:1 primarily activates rather glial cells than neurons, which may subsequently modulate neuronal responsiveness and sensory sensations such as itch and pain.     

Development of the Schwann cell lineage: from the neural crest to the myelinated nerve

The myelinating and nonmyelinating Schwann cells in peripheral nerves are derived from the neural crest, which is a transient and multipotent embryonic structure that also generates the other main glial subtypes of the peripheral nervous system (PNS). Schwann cell development occurs through a series of transitional embryonic and postnatal phases, which are tightly regulated by a number of signals. During the early embryonic phases, neural crest cells are specified to give rise to Schwann cell precursors, which represent the first transitional stage in the Schwann cell lineage, and these then generate the immature Schwann cells. At birth, the immature Schwann cells differentiate into either the myelinating or nonmyelinating Schwann cells that populate the mature nerve trunks. In this review, we will discuss the biology of the transitional stages in embryonic and early postnatal Schwann cell development, including the phenotypic differences between them and the recently identified signaling pathways, which control their differentiation and maintenance. In addition, the role and importance of the microenvironment in which glial differentiation takes place will be discussed.     

Fibroblast Growth Factors and Insulin Growth Factors Combine to Promote Survival of Rat Schwann Cell Precursors Without Induction of DNA Synthesis

In embryonic rat nerves, we recently identified an early cell in the Schwann cell lineage, the Schwann cell precursor. We found that when these cells were removed from contact with axons they underwent rapid apoptotic death, and that in a proportion of the cells this death could be prevented by basic fibroblast growth factor (bFGF, FGF-2). We now report that 100% of Schwann cell precursors isolated from peripheral nerves of 14-day-old-rat embryos can be rescued by a combination of insulin-like growth factor (IGF) 1 or 2 in combination with either acidic FGF (aFGF, FGF-1), bFGF or Kaposi's sarcoma FGF (K-FGF; FGF-4). The precursors display an absolute requirement for both an IGF and an FGF to achieve maximal survival. Elevation of intracellular levels of cAMP by forskolin does not result in a significant shift in the IGF/FGF dose-response curves. In contrast, the percentage of precursors rescued by FGF in the presence of insulin is dramatically increased by elevation of cAMP. These growth factor combinations did not stimulate DNA synthesis significantly in Schwann cell precursors. These findings show that cooperation between growth factors is required to suppress cell death in Schwann cell precursors, and suggest that survival and DNA synthesis are regulated by distinct growth factor combinations in these cells. The observations are consistent with the idea that survival regulation by FGFs and IGFs plays an important role in the development of glial cells in early embryonic nerves.     

Colonization of the bowel by the precursors of enteric glia: studies of normal and congenitally aganglionic mutant mice

The terminal portion of the ls/ls mouse is congenitally aganglionic because the precursors of enteric neurons fail to enter this region. This animal was studied in order to gain insight into the origin of enteric glia and into the process by which the precursors of these cells colonize the gut. In control (CD-1) mice, immunoreactivity of the glial marker, glial fibrillary acidic protein, appeared for the first time in the fetal bowel at day E16 and, in adults, was much more intense within intraenteric neural elements than in nerves outside the bowel. Glial fibrillary acidic protein developed in tissue cultures of fetal intestine explanted before the protein appeared in situ, and before the bowel became innervated by extrinsic nerves; thus, the precursors of cells able to elaborate glial fibrillary acidic protein must have been present, but unrecognizable, in the original explants. This explant assay demonstrated that these glial precursors were present in all regions of the bowel of control mice, but not in the presumptive aganglionic bowel of ls/ls mice. The nerves (of extrinsic origin) in the aganglionic tissue of ls/ls mice showed a high level of immunoreactive glial fibrillary acidic protein; nevertheless, their ultrastructure was typical of peripheral nerve, not enteric plexus, and they contained Schwann cells, not enteric glia. These observations support the view that enteric glia are derived from the single wave of neural crest colonists that populates the enteric nervous system before the gut receives its extrinsic innervation. These glial precursors, like neuronal precursors, tend to be excluded from the presumptive aganglionic ls/ls bowel. In contrast, Schwann cells grow into the abnormal ls/ls gut with the extrinsic innervation. The enteric microenvironment appears to promote the expression of glial fibrillary acidic protein in both enteric glia and Schwann cells; however, even within the bowel, Schwann cells retain their characteristic morphology. It is thus probable that the normal enteric nervous system contains supporting cells of separate lineages, enteric glia and Schwann cells.     

SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS

SPARC-like 1 (SC1) is a member of the SPARC family of matricellular proteins that has been implicated in the regulation of processes such as cell migration, proliferation, and differentiation. Here we show that SC1 exhibits remarkably diverse and dynamic expression in the developing and adult nervous system. During development, SC1 localizes to radial glia and pial-derived structures, including the vasculature, choroid plexus, and pial membranes. SC1 is not downregulated in postnatal development, but its expression shifts to distinct time windows in subtypes of glia and neurons, including astrocytes, large projection neurons, Bergmann glia, Schwann cells, and ganglionic satellite cells. In addition, SC1 expression levels and patterns are not altered in the SPARC null mouse, suggesting that SC1 does not compensate for the absence of SPARC. We conclude that SC1 and SPARC may share significant homology, but are likely to have distinct but complementary roles in nervous system development.     

Inhibition of glial maturation by bone morphogenetic protein 2 in a neural crest-derived cell line

Bone morphogenetic proteins (BMPs) regulate developmental decisions in many neural and nonneural lineages. BMPs influence both CNS neuronal and glial development and promote neuronal differentiation in neural crest derivatives. We investigated the actions of BMP2 on glial differentiation in the peripheral nervous system using NCM1 cells, a neural crest-derived cell line with the properties of peripheral glial precursor cells. BMP2 prevented the acquisition of a mature Schwann cell-like morphology, blocking the expression of mature genes and maintaining expression of several early glial markers. We provide evidence that BMP2 activates the GFAP promoter and define signaling pathways underlying this regulation. Our results demonstrate a novel role for BMPs as inhibitors of glial differentiation in the peripheral nervous system and suggest that BMPs may regulate the developmental timing of glial maturation.     

Spatiotemporal distribution and function of N‐cadherin in postnatal Schwann cells: A matter of adhesion?

During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N‐cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N‐cadherin expression levels decrease as precursors differentiate into Schwann cells. In this study, we investigated the distribution of N‐cadherin in the developing postnatal and adult rat peripheral nervous system. N‐cadherin was found primarily in ensheathing glia throughout development, concentrated at neuron–glial or glial–glial contacts of the sciatic nerve, dorsal root ganglia (DRG), and myenteric plexi. In the sciatic nerve, N‐cadherin decreases with age and progress of myelination. In adult animals, N‐cadherin was found exclusively in nonmyelinating Schwann cells. The distribution of N‐cadherin in developing E17 DRG primary cultures is similar to what was observed in vivo. Functional studies of N‐cadherin in these cultures, using the antagonist peptide INPISGQ, show a disruption of the attachment between Schwann cells, but no interference in the initial or long‐term contact between Schwann cells and axons. We suggest that N‐cadherin acts primarily in the adhesion between glial cells during postnatal development. It may form adherents/junctions between nonmyelinating glia, which contribute to the stable tubular structure encapsulating thin caliber axons and thus stabilize the nerve structure as a whole. © 2010 Wiley‐Liss, Inc.     

Oligodendrocyte and astrocyte development in rodents: an in situ and immunohistological analysis during embryonic development

Lineally related multipotent neuroepithelial cells (NEP), neuronal restricted precursors (NRP), and glial restricted precursors (GRP) have been identified in the spinal cord. To determine the sequence of differentiation and identify lineage and stage-specific markers, we have examined the spatiotemporal expression of established glial markers during rodent embryonic development and within fetal cell culture. In this report, we show that proliferating stem cells in the developing neural tube do not express any glial markers at E10.5. By E11, however, glial precursors have begun to differentiate and at least two regions of the ventral neural tube containing glial precursor cells can be distinguished, an Nkx2.2/Neurogenin 3 (Ngn3) domain and a platelet-derived growth factor receptor alpha (PDGFRalpha)/Olig2/Sox10 domain. Radial glia, as identified by RC1 immunoreactivity, develop in concert with other glial precursors and can be distinguished by their morphology, spatial distribution, and antigen expression. Astrocytes as assessed by glial fibrillary acidic protein (GFAP) immunoreactivity are first detected at E16. A novel dorsal domain of CD44 immunoreactivity that can be distinguished from the more ventral glial precursor domains can be detected as early as E13.5.     

In vitro and in vivo differentiation of boundary cap neural crest stem cells into mature Schwann cells

Boundary cap cells can generate neurons as well as peripheral glia during embryonic development (Maro, G.S., Vermeren, M., Voiculescu, O., Melton, L., Cohen, J., Charnay, P., Topilko, P., 2004. Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS. Nat Neurosci. 7 (9), 930-938), and, recently, the boundary cap was shown to contain multipotent stem cells (Hjerling-Leffler, J., Marmigère, F., Heglind, M., Cederberg, A., Koltzenburg, M., Enerb?ck, S., Ernfors, P., 2005. The boundary cap, a source of neural crest stem cells generating multiple sensory neuron subtypes. Development. 132 (11), 2623-2632). The ability of stem cells to generate mature functional glial phenotypes has not been addressed. In this study, we have explored the competence of boundary neural crest stem cells (bNCSCs) to differentiate into mature functional Schwann cells (SCs) in vitro and in vivo. bNCSCs failed to differentiate into SCs in vitro when cultured in a defined media and in vivo when grafted into adult rat sciatic nerves. However, in the presence of neuregulins, during long-term cultures, the majority of bNCSCs differentiated into SCs. After analysis of the in vivo expression of Sox2, Sox10, S100, GFAP, fibronectin and Krox20 in the glial lineages, we used these markers to characterize differentiation of the bNCSCs. Gliogenesis of bNCSCs proceeded similar to that in vivo by sequentially adopting a SC precursor and immature Schwann cell before maturing into myelinating and non-myelinating SCs. In co-culture with explanted dorsal root ganglia (DRG) as well as in vivo in transplants to the axotomized sciatic nerve, these bNCSC-derived SCs myelinated axons as shown by ensheathing of neuronal processes and expression of myelin basic proteins (MBP). These results show that, under appropriate conditions, bNCSCs can generate mature SCs that are functional and can myelinate axons in regenerating nerves.     

Involvement of 5-HT7 receptors in serotonergic effects on spike afterpotentials in presumed jaw-closing motoneurons of rats

Supporting glial cells of the peripheral nervous system include satellite cells of dorsal root ganglia and Schwann cells of peripheral nerves. In the central nervous system, glial cells contain enzymes related to the tricarboxylic acid and glutamine cycles: pyruvate carboxylase, glutamate dehydrogenase, and glutamine synthetase. The present study used immunohistochemistry in the rat peripheral nervous system to determine the cellular distribution of these enzymes along with glutamine. In dorsal root ganglia and peripheral nerves, glutamine and glutamine related enzymes were enriched in satellite and Schwann cells. In the dorsal root ganglia, immunoreactive satellite cells surrounded neurons of all sizes. In peripheral nerve, immunoreactive Schwann cells were most easily observed surrounding large diameter, myelinated axons. These Schwann cells contained immunoreactivity in their cell bodies, nodes of Ranvier, and the rim of cytoplasm outside the myelin sheath. Myelin sheaths were non-immunoreactive. The peripheral glial tricarboxylic and glutamine cycles may be used to produce glutamine for neuronal cell uptake and conversion to glutamate for synaptic transmission. Alternatively, these cycles may function in peripheral glia similar to central nervous system astrocytes for supporting the energy demands of neurons.     

Schwann cell development in embryonic mouse nerves.

Previously we proposed that Schwann cell development from the neural crest is a two-step process that involves the generation of one main intermediate cell type, the Schwann cell precursor. Until now Schwann cell precursors have only been identified in the rat, and much remains to be learned about these cells and how they generate Schwann cells. Here we identify this cell in the mouse and analyze its transition to form Schwann cells in terms of timing, molecular expression, and extracellular signals and intracellular pathways involved in survival, proliferation, and differentiation. In the mouse, the transition from precursors to Schwann cells takes place 2 days earlier than in the rat, i.e., between embryo days 12/13 and 15/16, and is accompanied by the appearance of the 04 antigen and the establishment of an autocrine survival circuit. Beta neuregulins block precursor apoptosis and support Schwann cell generation in vitro, a process that is accelerated by basic fibroblast growth factor 2. The development of Schwann cells from precursors also involves a change in the intracellular survival signals utilized by neuregulins: To block precursor death neuregulins need to signal through both the mitogen-activated protein kinase and the phosphoinositide-3-kinase pathways although neuregulins support Schwann cell survival by signaling through the phosphoinositide-3-kinase pathway alone. Last, we describe the generation of precursor cultures from single 12-day-old embryos, a prerequisite for culture studies of genetically altered precursors when embryos are non-identical with respect to the transgene in question.     

The olfactory nerve contains two populations of glia, identified both in vivo and in vitro.

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.     

Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia

The similarities between the enteric nervous system of the gut and the central nervous system (CNS), both of which function as complex integrative nervous networks, include striking ultrastructural similarities between the glia of the enteric nervous system and the astrocytic glia of the CNS. In this paper we have determined whether this anatomical resemblance also extends to the molecular level by examining the enteric glial cells to see whether they express several surface and intracellular molecules which are highly restricted to glia and to astrocytes in particular. Indirect immunofluorescence was used to visualize the antigens in frozen sections of gut wall and in whole mount, tissue culture, and freshly dissected preparations of myenteric and submucous plexuses from rats of various ages. It was found that enteric glial cells expressed the intracellular proteins glial fibrillary acidic protein, glutamine synthetase, and vimentin both in situ and in culture. The surface antigen Ran-2 was expressed in situ but not in culture, and the surface antigen Ran-1 was expressed in culture but not in situ. Cultured enteric glial cells did not express fibronectin in significant quantity, nor did they make galactocerebroside. From these results we conclude that the adult phenotype of enteric glia in situ closely resembles that of astrocytes, while in culture some of their cell surface features change, reverting to those seen during development. Because these cells possess distinctive molecular features and numerically form one of the major populations of peripheral glia, it is appropriate to classify them as a third distinctive category of peripheral glial cells, in addition to satellite and Schwann cells. The molecular similarities between these cells and astrocytes, in addition to their anatomical resemblance, suggest that a further study of enteric glia will provide new insights into the role of glia in integrative nervous tissues.     

Radial glia phenotype: origin, regulation, and transdifferentiation

Radial glial cells play a major guidance role for migrating neurons during central nervous system (CNS) histogenesis but also play many other crucial roles in early brain development. Being among the earliest cells to differentiate in the early CNS, they provide support for neuronal migration during embryonic brain development; provide instructive and neurotrophic signals required for the survival, proliferation, and differentiation of neurons; and may be multipotential progenitor cells that give rise to various cell types, including neurons. Radial glial cells constitute a major cell type of the developing brain in numerous nonmammalian and mammalian vertebrates, increasing in complexity in parallel with the organization of the nervous tissue they help to build. In mammalian species, these cells transdifferentiate into astrocytes when neuronal migration is completed, whereas, in nonmammalian species, they persist into adulthood as a radial component of astroglia. Thus, our perception of radial glia may have to change from that of path-defining cells to that of specialized precursor cells transiently fulfilling a guidance role during brain histogenesis. In that respect, their apparent change of phenotype from radial fiber to astrocyte probably constitutes one of the most common transdifferentiation events in mammalian development.     

Schwann cells and astrocytes induce synapse formation by spinal motor neurons in culture

Glia constitute 90% of cells in the human nervous system, but relatively little is known about their functions. We have been focusing on the potential synaptic roles of glia in the CNS. We recently found that astrocytes increase the number of mature, functional synapses on retinal ganglion cells (RGCs) by sevenfold and are required for synaptic maintenance in vitro. These observations raised the question of whether glia similarly enhance synapse formation by other neuron types. Here we have investigated whether highly purified motor neurons isolated from developing rat spinal cords are able to form synapses in the absence of glia or whether glia similarly enhance synapse number. We show that spinal motor neurons (SMNs) form few synapses unless Schwann cells or astrocytes are present. Schwann cells increase the number of functional synapses by ninefold as measured by immunostaining, and increase spontaneous synaptic activity by several hundredfold. Surprisingly, the synapses formed between spinal motor neurons were primarily glutamatergic, as they could be blocked by CNQX. This synapse-promoting activity is not mediated by direct glial-neuronal cell contact but rather is mediated by secreted molecule(s) from the Schwann cells, as we previously found for astrocytes. Interestingly, the synapse-promoting activity from astrocytes and Schwann cells was functionally similar: Schwann cells also promoted synapse formation between retinal ganglion cells, and astrocytes promoted synapse formation between spinal motor neurons. These studies show that both astrocytes and Schwann cells strongly promote synapse formation between spinal motor neurons and demonstrate that glial regulation of synaptogenesis extends to other neuron types.