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Neurodegenerative effects of Schwann cells transplanted into the central nervous system have been observed previously. We report here that conditioned medium from Schwann cell cultures exhibit degenerative influences on hippocampal neurons. Aliquots of Schwann cell-conditioned medium compromised the morphologic integrity of the neurons, markedly elevated their intracellular calcium concentrations, and decreased their viability. The degenerative effects of Schwann cell medium on neuronal morphology and viability were blocked by N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonopentanoic acid (D-APV) and 5,7-dicholorokynurenic acid (DCKA). Glutamate was detected in Schwann cell-conditioned medium at a concentration on the order of 10(-5) M. D-Amino acid oxidase (DAAOx) also attenuated the neurotoxicity exhibited by Schwann cells. These data suggest that Schwann cells release biologically relevant concentrations of excitotoxins that include glutamate and D-serine.

D-serine appears to be a natural agonist at the "glycine site" of the NMDA receptor and is created by conversion from L-serine by serine racemase. This racemase has been localized to protoplasmic astrocytes that ensheath synapses and modulate neuronal activity in the CNS, but serine racemase expression in the PNS has not been reported. Immunofluorescence indicated that Schwann cells and other endoneurial components of rat spinal nerve contain serine racemase, and western blot analysis detected the enzyme in lysates of sciatic nerve. Cultures from sciatic nerve contained Schwann cells and fibroblasts, and both cell types showed serine racemase expression by immunofluorescence and Western blot; the quantities per unit of total protein appeared slightly lower than that expressed in cultured astrocytes. Cultures enriched for each cell type were subjected to reverse transcriptase polymerase chain reaction, further confirming serine racemase mRNA in Schwann cells and fibroblasts. Finally, immunodetection of D-serine itself was observed in cultured Schwann cells and fibroblasts. These expression patterns of serine racemase may indicate roles for D-serine in peripheral nerve transduction.

This study examines a radiation-induced invasion and spread of Schwann cells into ventral gray regions of the lumbar spinal cord. The prevalence of these cells within the gray matter and the time course of their appearance in the ventral spinal cord is quite different from the pattern of Schwann cell development in dorsal spinal cord reported previously. The focus is on 2 possible pathways, each involving astrocytic barriers, by which Schwann cells access the ventral gray matter. The first of these is the glia limitans covering the ventral surface of the spinal cord and the possibility that its integrity has been disrupted by the exposure to x-rays. Comparisons of the glia limitans, including its thickness, between irradiated and nonirradiated rats revealed that exposure to radiation did not result in any morphologically discernible alterations. The second barrier examined was the astrocytic covering of blood vessels. In irradiated animals the astrocyte processes that normally surround blood vessels were missing in some instances, and Schwann cells were observed at these sites. The difference between the dorsal and ventral occurrence of Schwann cells is that, whereas Schwann cells primarily follow axons, specifically dorsal root axons, to access the dorsal spinal cord, it appears that the presence of Schwann cells in the ventral portion of the spinal cord where their location is primarily in the gray matter is associated with the vasculature.

This review summarises some of the major findings derived from studies using the model of a glia-depleted environment developed and characterised in this laboratory. Glial depletion is achieved by exposure of the immature rodent spinal cord to x-radiation which markedly reduces both astrocyte and oligodendrocyte populations and severely impairs myelination. This glia-depleted, hypomyelinated state presents a unique opportunity to examine aspects of spinal cord maturation in the absence of a normal glial population. An associated sequela within 2–3 wk following irradiation is the appearance of Schwann cells in the dorsal portion of the spinal cord. Characteristics of these intraspinal Schwann cells, their patterns of myelination or ensheathment, and their interrelations with the few remaining central glia have been examined. A later sequela is the development of Schwann cells in the ventral aspect of the spinal cord where they occur predominantly in the grey matter. Characteristics of these ventrally situated intraspinal Schwann cells are compared with those of Schwann cells located dorsally. Recently, injury responses have been defined in the glia-depleted spinal cord subsequent to the lesioning of dorsal spinal nerve roots. In otherwise normal animals, dorsal nerve root injury induces an astrocytic reaction within the spinal segments with which the root(s) is/are associated. Lesioning of the 4th lumbar dorsal root on the right side in irradiated or nonirradiated animals results in markedly different glial responses with little astrocytic scarring in the irradiated animals. Tracing studies reveal that these lesioned dorsal root axons regrow rather robustly into the spinal cord in irradiated but not in nonirradiated animals. To examine role(s) of glial cells in preventing this axonal regrowth, glial cells are now being added back to this glia-depleted environment through transplantation of cultured glia into the irradiated area. Transplanted astrocytes establish barrier-like arrangements within the irradiated cords and prevent axonal regrowth into the cord. Studies using other types of glial cultures (oligodendrocyte or mixed) are ongoing.

Schwann cells develop within the ventral gray matter following exposure of lumbosacral spinal cords to x-rays in early postnatal rats. These ventral gray matter Schwann cell aggregates occurred in about 40% of the animals 8 or more weeks following irradiation. Light microscopically these cells appeared to be apposed to somata of large motor neurons, raising a question regarding the fate of axo-somatic synapses. This study focused on neuron-Schwann cell relationships and demonstrated ultrastructurally that the intraspinal Schwann cells established a variety of relationships with the neuronal somata and primary dendrites. These relationships ranged from direct contact without an intervening basal lamina to the presence of synaptic contacts intervening between neuron and Schwann cell basal lamina. Occasionally, the Schwann cells occupied an intermediate position between neurons and blood vessels, suggesting functions similar to those carried out by astrocytes. In these instances, as in all cases of Schwann cell-blood vessel contact, the vessels lacked their normal investiture by astrocytes. Light microscopic evaluation of synaptophysin-immunostained sections revealed decreased immunoreactivity in neuropil occupied by the Schwann cells but confirmed the presence of synapses on neuronal somata. Possible mechanisms underlying Schwann cell induction in the ventral gray matter are discussed. An understanding of the interactions between Schwann cells and the cellular constituents of the gray matter is important in light of attempts to enhance repair in the central nervous system by transplanting Schwann cells into that environment. © 1996 Wiley-Liss, Inc.

The glial population of the lumbosacral spinal cord of the rat can be markedly depleted by exposure to ionizing radiation during the first postnatal week. Identification of specific cell populations which survive the exposure to radiation is difficult in situ; therefore, the present investigation used in vitro approaches to address issues related to specific phenotypes and maturational states of glia in cultures derived from non-irradiated (control) and irradiated (experimental) lumbosacral spinal cords of 3-day-old rats. Cultures were established from the spinal cords 2 to 4 hours following irradiation and were compared to cultures from non-irradiated, littermate controls. By 4 days in vitro (DIV) the numbers of cells in experimental cultures were profoundly reduced when compared to controls, and this reduction persisted through the termination of the study (8 DIV). In addition to reduction in numbers, astrocyte phenotypes were altered in experimental cultures, with greater proportions of the astrocyte population being constituted by the flat angular, large angular, and pancake types and a lesser proportion by stellate cells. The non-astrocytic cell types were dramatically reduced as evidenced by the paucity of oligodendrocytes immunoreactive for galactocerebroside and of small, non-process bearing cells binding the lectin, Griffonia (Bandeiraea) simplicifolia, a marker for microglia. Experimental cultures contained an increased incidence of binucleate astrocytes, an increase not restricted to a particular astrocyte phenotype. This study established the feasibility of utilizing this combined in vivo/in vitro approach in assessment of glial populations in immature spinal cords, and further investigations are in progress using this model.

Exposure of the rat lumbar spinal cord to X-rays during the early postnatal period results in a marked reduction in the glial populations within the irradiated region. The present study was undertaken to determine what effects this reduction of glia, particularly astrocytes, has on the pattern and characteristics of the scar formation that follows root injury in the normal spinal cord. Morphological assessments 60 days following injury of the right L4 dorsal root revealed a distinct difference in the extent of the astrocyte response between the irradiated and the nonirradiated rats. In the nonirradiated animals, a thick astrocytic scar composed of multiple layers of astrocyte processes formed over the dorsal horn and adjacent portions of the dorsal surface of the cord. This astrocytic response was not confined to the surface of the spinal cord but extended also into the root, i.e., into regions normally considered as PNS. In irradiated rats, the astrocytes did not form a thick scar nor did they extend into the injured root. Instead, they formed a glia limitans and were at most only one or two layers thick over the region of cord comparable to that occupied by the thick astrocytic scar in the nonirradiated rats. Mechanisms involved in the glial response of the irradiated spinal cord to dorsal root injury are discussed, particularly with regard to the possible positive effect that this reduction in scar formation may have on regrowth of injured dorsal root axons into the spinal cord environment.

In order to investigate axolemmal development in a glial cell deficient environment, normal and irradiated dorsal funiculus in rat lumbosacral spinal cord was examined by freeze-fracture electron microscopy. At 3 days of age, normal fibres are all unmyelinated and of small (less than 0.5 micron) diameter. The unmyelinated axons have a moderate density (approximately 850 microns-2) of intramembranous particles (IMPs) on P-fracture faces and a low IMP density (approximately 300 microns-2) on E-faces. IMPs are homogeneously distributed along both fracture faces. By 19 days of age, the normal dorsal funiculus is well populated with myelinated axons and glial cells, as well as a sizable population of unmyelinated fibres. Nearly all of the myelinated fibres have a large (greater than 1.0 micron) diameter; whereas, most unmyelinated axons are of small (less than 0.5 micron) calibre. The axolemma of unmyelinated axons is relatively undifferentiated, with an asymmetrical distribution of IMPs (P-face: approximately 1100 microns-2; E-face: approximately 450 microns-2). Myelinated fibres show nodal and paranodal regions with P-face and E-face ultrastructure similar to previous descriptions. Internodal axolemma appears relatively homogeneous, with P-faces being highly particulate (approximately 2100 microns-2) and a low IMP density (approximately 200 microns-2) on E-faces. Following irradiation of the lumbosacral spinal cord at 3 days of age, there is a severe reduction in the number of glial cells and myelinated fibres in this region when the tissue is examined at 19 days of age. Despite the deficiency of glial cells in this tissue, axonal and axolemmal development continue. Numerous large (greater than 1.0 micron) diameter axons are present in this irradiated tissue. Large diameter axons show a high (approximately 2000 microns-2) density of IMPs on P-faces; E-face IMP density remains at approximately 440 micron-2. Small calibre axons also have an asymmetrical distribution of particles (P-face: approximately 1100 microns-2; E-face: 280 microns-2). The axolemmal E-faces of some glial cell deprived fibres exhibit regions with greater than normal (approximately 750 microns-2) density of IMPs. These results demonstrate that some aspects of axonal and axolemmal development continue in a glial cell deficient environment, and it is suggested that axolemmal ultrastructure is, at least in part, independent of glial cell association.

Tritiated thymidine autoradiography has been used to study the generation of lateral motor neurons (LMNs), association interneurons (ANs) and dorsal root ganglion cells (DRGNs) in the spinal cords of genetically diverse strains of mice. The neuronal populations analyzed in this study form an early developing reflex pathway and the ontogeny of this circuit exhibits genetically associated variability. The strains of mice used in this investigation have been shown to differ in the embryonic age at which forelimb reflex movements are first manifest and in the timing of synapse formation within the reflex pathway. A precocious development of these reflex traits occurs in strain C57BL/6J in comparison to embryos of intermediate (CBA/CaJ) and late developing (LP/J) strains. All three inbred strains show the same basic generation sequence for the neuronal populations comprising the forelimb reflex pathway. The generation of LMNs precedes that of ANs, and the generation of ANs, in turn, precedes that of DRGNs. Since this is the same sequence as that observed for the formation of synaptic junctions in all three strains, it is suggested that synaptogenic sequences in reflex circuits may be determined by the generation sequence of the component neuronal populations. Although the strains all exhibit the same basic sequence of neuronal generation, the temporal relationships of the generation of each cell population within this sequence show significant strain dependent variations. C57BL/6J displays a larger temporal separation between the generation of each cell type than LP/J, and CBA/CaJ is intermediate to the other two strains in this respect. The fact that this strain order is identical to that observed for the development of reflex traits suggests that genetically associated differences in the timing of neuronal birthdays within a common generation sequence may have a substantial influence on the timing of synaptogenesis within the reflex pathway.

Axonal growth cones and developing axonal collaterals have been studied in electron microscopic and Golgi preparations of embryonic mouse cervical spinal cord. These structures are first observed on embryonic day 11 (E11), and by E12 both axonal growth cones and developing collaterals are observed to be the presynaptic elements of developing synaptic junctions. These relatively undifferentiated portions of axons comprise nearly one-half of the presynaptic components of the synaptic contacts observed on E12, and they continue to constitute about this same proportion during the rest of the embryonic period examined (E13–16). These early synaptic interactions of developing axonal collaterals may be involved in establishing the basic ramification patterns of mature axons by determining which developing collaterals will continue to grow and differentiate. Coated vesicles are observed fused with axonal collateral plasma membranes at the perimeters of developing synaptic contacts. Occasionally thin extensions of postsynaptic surfaces protrude into these coated vesicles, and similar relationships are also observed between presynaptic surfaces and coated vesicles fused with the membranes of postsynaptic elements. Such coated vesicle/ synaptic surface relationships may represent an early stage in the endocytosis of synaptic membranes by both the pre- and postsynaptic elements of developing synaptic contacts. This might represent a mechanism for the exchange of ‘molecular information’ between the components of protosynaptic contacts, and the results of such an exchange could be involved in determining whether protosynaptic contacts will differentiate into synaptic junctions. Developing axonal collaterals exhibit a preferential growth toward the sources of potential postsynaptic elements, and this growth implies that the axons present in the embryonic marginal zone are actively searching for appropriate postsynaptic processes rather than passively awaiting their arrival. Many of the collaterals weave around numerous intervening processes in order to reach their postsynaptic destinations, and unusual subaxolemmal lamellae are commonly observed where collaterals make acute curvatures around neurites which block their direct access to postsynaptic processes. The location of these lamellae suggests that they could be involved in producing changes in the direction of growth as developing axonal collaterals search for synaptic partnerships. Main axonal shafts commonly exhibit synaptic contacts adjacent to where they give rise to developing collaterals, and this observation hints that the formation of synapses by axonal shafts might stimulate collateral sprouting. However, other observations (see text) indicate that the formation of synaptic contacts by axonal shafts is not an obligatory prelude to collateral development. Therefore synaptic interactions of main axonal shafts do not appear to be a requirement for the primary induction of collateral development, but they may facilitate collateral growth by providing a confirmation that axons are located in appropriate synaptogenic fields.

The ultrastructure of paranodal oligodendroglial cytoplasm, which is located in proximity to the forming myelin sheath, was studied during maturation of spinal motoneuron axons in rat. At 8 days postnatal, the paranodal oligodendroglial loops contain a network of membrane-bound tubulovesicular elements. These membrane elaborations are most common in oligodendroglial loops attached to the outermost layers of the myelin sheath, i.e., paranodal loops closest to the nodal gap. The number of oligodendroglial cytoplasmic profiles per paranodal loop falls over the course of five to ten sequential paranodal loops, and these profiles are nearly absent in paranodal oligodendroglial cytoplasm located distant from the nodal gap. Oligodendrocytes in spinal cords of 14- and 20-day-old rats and of adult rats did not exhibit networks of tubulovesicular profiles. The appearance of these membrane organelles within oligodendroglial cytoplasm during myelin maturation suggests increased membrane turnover within paranodal cytoplasm located adjacent to the axon that is being myelinated. Membrane turnover within oligodendrocytes may reflect axonal modulation of glial function during myelination.

Branching patterns of dendrites may be modulated by the way in which dendritic growth cone filopodia come into initial synaptic relationships with afferent axons. This synaptotropic hypothesis of dendritic branching predicts that dendritic growth will be directed preferentially into regions containing numerous prospective presynaptic elements. The developing mouse spinal cord provides a natural experiment to test this prediction, because synapses are found exclusively within the marginal zones bordering the motor columns during the early (E11-14) period of synaptogenesis. During this time, therefore, most motor dendritic growth would be expected to be directed laterally or ventrally into the marginal zones, whereas internally directed growth should become more prevalent later, when synaptogenesis begins to take place within the intermediate zone, i.e., the motor columns proper. A computer-assisted three dimensional reconstruction system has been used to test these expectations in Golgi preparations of developing mouse (C57BL/6J) spinal cords ranging in age from E13 through P1. Mean dendritic lengths and branch densities are significantly greater for marginal zone dendrites than for intermediate zone dendrites at early ages (E13-14), but there are no significant differences in these measures at later stages of development (P0,1). These findings are interpreted as meaning that motor dendritic growth is initially biased into the marginal zone by synaptogenic afferents and that this preferential distribution is progressively lost as synapses develop within the intermediate zone to attract or to stabilize internally directed dendritic growth. Thus the findings of this study are consistent with predictions of the synaptotropic hypothesis of dendritic branching.