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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.

Synaptophysin immunostaining of areas of spinal gray matter occupied by radiation-induced intraspinal Schwann cells revealed a loss of immunoreactivity from the neuropil. In contrast, synaptophysin immunoreactivity was preserved on the somata and proximal dendrites of motor neurons. The present study extended these observations to the ultrastructural level and confirmed the absence not only of synapses but also of astrocytes and small- and medium-sized dendrites. These neural elements were abundant and appropriately organized in contiguous areas of irradiated neuropil not occupied by Schwann cells.

Patterns of synaptophysin immunoreactivity were examined in the ventral and lateral funiculi of rat lumbosacral spinal cords. In normal young adults, dendrites from neurons in the spinal gray matter extended into the ventral and lateral white matter as finger-like projections, immunopositive for synaptophysin. These projections appeared to diminish in size as they extended peripherally and, in general, did not reach the surface of the spinal cord, so that the outer one-third to one-fourth of the funiculi contained little or no immunoreactivity. The spinal cords of some of the animals studied were X-irradiated on the third postnatal day. When examined 6 weeks to 5 months later, the pattern of synaptophysin immunoreactivity was found to be markedly altered in these animals. In general, the synaptophysin immunoreactivity in the white matter was less organized than in the non-irradiated rat. As a result, the finger-like projections, particularly into the lateral funiculi, were not as distinct, and the immunoreactivity appeared to be more diffusely distributed in the white matter. Further, the immunoreactivity was present throughout the thickness of the white matter in the irradiated animals and subpial concentrations were evident, especially along the lateral aspect of the spinal cord. Ultrastructural evaluation of the synaptic profiles revealed no differences between irradiated and non-irradiated animals. The synapses occurred on both the shafts of the dendrites and on the spines. In general, both dendrites and axon terminals were covered by astrocyte processes except at synaptic sites, and the synaptic complexes were surrounded by astrocyte processes. Although the mechanisms underlying the altered pattern of synaptophysin immunoreactivity are not yet understood, they may be related to radiation-induced effects on the glial populations previously reported by the investigators and/or to radiation-induced alterations in reorganization or maturation of dendritic trees.

Studies of potentials for dorsal spinal nerve root axons to regrow into the spinal cord involved placement of the tracer HRP/WGA-HRP on the cut end of the nerve root. Following this procedure, labeled neurons were found within the spinal dorsal gray matter. Analyses revealed that spinal neurons influenced by the presence of radiation-induced intraspinal Schwann cells extend misdirected processes into the dorsal root.

Previous studies in this laboratory have documented that the microglial environment of the immature spinal cord is altered by exposure to ionizing radiation. As a result, the lumbosacral spinal cord is markedly depleted of both oligodendrocytes and astrocytes, while leaving axons and the overall cytoarchitecture intact. The status of the microglia in the irradiated region is unknown and is of interest given the interactions between microglia and astrocytes recently elucidated by others. This study uses both in vivo and in vitro approaches to examine the microglial population in normal and irradiated immature spinal cord. The lectin, Griffonia (Bandeiraea) simplicifolia, was selected since it marks microglia both in paraffin embedded sections and in cell cultures. Light microscopic examinations of spinal cord sections revealed a reduced microglial population in the irradiated region when compared to littermate controls, and a change in morphology of the remaining microglia to that described by others as "activated". Cultures prepared from lumbosacral spinal cords harvested from 3-day-old rats within 2-4 hr following irradiation were compared with cultures derived from their non-irradiated littermates after 8 days in vitro. Cultures from the irradiated spinal cords revealed trends similar to those observed in vivo, i.e. a reduced microglial population and altered morphology. Although all glial cell types were reduced in cultures from irradiated spinal cords, the few microglia present were usually positioned atop astrocytes. The consistency of reduction in all glial populations in this model shows the microglia to be a novel microenvironment for further studies of roles of microglial within the spinal cord.

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.

Exposure of the lumbosacral spinal cord of early postnatal rats to X-rays reduces the glial populations within the irradiated region. The present study examines the ability of axons of a dorsal root subjected to a crush-freeze lesion to grow back into this glial-deficient spinal cord environment, in contrast to the non-irradiated rat. Ultrastructural examination of the dorsal root entry zone (DREZ) 60 days after root injury revealed a well-formed astrocytic scar in this zone and adjacent regions of spinal cord in non-irradiated rats. In contrast, scar formation did not occur in irradiated root-lesioned animals in which the astrocytic response was quite limited. Axons were present in the DREZ and underlying spinal cord in irradiated root-lesioned rats at this time but were absent from these regions in the non-irradiated lesioned controls. These ultrastructural findings are highly suggestive that axons are capable of regrowth into the irradiated spinal cord. Axonal regrowth was assessed further by tracing techniques after application of a combination of peroxidase-labeled wheat germ agglutinin and horseradish peroxidase to the cut end of the root distal to the previously injured site. Labeled axons were readily identified within the spinal gray matter in irradiated lesioned but not in the non-irradiated lesioned rats. These data, together with the ultrastructural observations, are supportive of regrowth of the dorsal root axons into the spinal cord. The radiation-induced changes in the glial populations are discussed with regard to conversion of a normally non-permissive environment into one conducive for axonal regrowth.

Nodes of Ranvier in the central nervous system in mammals are characterized by the presence of perinodal astrocytic processes. This study examines the association between processes of radial glia and the axolemma at nodes of Ranvier in the spinal cord of the mature axolotl, an animal in which radial glia represent a large portion of the total glial population. The radial glial cells have their cell bodies located close to the central canal. Those situated dorsal to the canal send long processes to the dorsal surface of the spinal cord. Along this trajectory these processes coalesce into large fascicles in the midline and form the dorsal median septum. Slender branches rise from the processes in these fascicles and extend into the adjacent white matter to terminate in close apposition to the axolemma at nodes of Ranvier. This arrangement provides an intracellular pathway extending from the perinodal region to the surface of the spinal cord. Radial glia ventral to the central canal give rise to processes that project to the glia limitans adjacent to the ventral spinal artery. These ventrally projecting processes appear to be more irregular in their branching pattern than their dorsal counterparts. Multiple slender processes are seen in close apposition to the nodal axolemma of myelinated axons in the ventral white commissure, again providing an intracellular pathway that runs from the perinodal region to the cord surface. In one instance a radial glial process was observed to occupy a pocket formed by the invagination of the nodal axolemma. The axonal cytoplasm adjacent to the invagination contained a variety of organelles, e.g. multivesicular bodies, vesicles and endoplasmic reticulum, suggesting that this relationship between the radial glial process and the axon is more than a passive interaction. These observations are consistent with the view that processes of radial glial cells may regulate the extracellular environment adjacent to the nodal axolemma, and/or play an active role in the maintenance of the nodal membrane. The existence of perinodally-directed processes of radial glial cells in the salamander indicates that this axo-glial specialization reflects an important functional interaction preserved across a large segment of the phylogenetic scale.

The major corticospinal tract (CST) in the rat is located at the base of the dorsal funiculus. It is a late-developing tract, and the growth of its axons into the lumbosacral region of the spinal cord does not occur until postnatal days 5 and 6. This delay is taken advantage of in this study in order to evaluate the effects of a markedly reduced glial population on ingrowth of the CST axons into the lumbosacral spinal cord. A reduction of the glial population is achieved by exposure of this region of spinal cord to X-radiation at 3 days of age. Growth of CST axons into and through the lumbosacral spinal cord in rats in which this region has undergone a radiation-induced depletion of glial cells is compared with that in their non-irradiated littermate controls by axonal tracing techniques using horseradish peroxidase (HRP). The HRP was applied directly to the motor cortices of normal and irradiated rats, and at all ages studied, there was anterograde filling of CST axons and their growth cones. At 3 days postnatally, the age when the lumbosacral spinal cord was irradiated in the experimental animals, CST axons were present in the more rostral thoracic levels. CST axons were observed in the lumbar region of non-irradiated rats on day 5, and by day 7 they were present at sacral levels. This same pattern was also noted in the animals with irradiated lumbosacral spinal cords: thus, despite the marked, radiation-induced reduction of glia in the region to be occupied and/or traversed by the ingrowing CST axons, the development of the tract appeared to be normal with respect to location and to time of arrival of the axons in the lumbosacral region.

Aging in the brain is associated with specific changes in the astrocyte population. The present study establishes that similar changes occur in the aging spinal cord. The levels of glial fibrillary acidic protein (GFAP) mRNA were significantly increased 0.4-fold in aged 8- to 17-month-old rats compared to young 2-month-old rats. The ability of astrocytes in the aging spinal cord to respond to a non-invasive CNS injury was compared to young rats 4 days following sciatic nerve axotomy. The level of GFAP mRNA was significantly increased 0.5-fold in the young rats in response to axotomy. In contrast, the level of GFAP mRNA in aged rats did not increase following injury above that present in non-axotomized rats of the same age.

The ventral funiculi of normal and X-irradiated 13-day-old rats were studied by electron microscopy. In both tissues, oligodendrocytes form myelin sheaths around multiple axons, with a single oligodendrocyte associated with several axons of different sizes. Despite their origin from the same glial cell, the myelin sheaths are thicker for larger axons. Polyribosomes and rough endoplasmic reticulum are observed in distal oligodendrocyte processes, in proximity to the forming myelin sheaths. These results indicate that myelin sheath thickness is matched to axon size via local mechanisms, and suggest a role of polyribosomes and/or rough endoplasmic reticulum in myelin formation.

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 spinal central canal area in the early postnatal rat differs markedly in appearance from that in the adult. Dorsally and ventrally, the walls of the canal lack nuclei and appear to be composed of processes from cell bodies at some distance from the lumen. Additionally, a densely packed cluster of cells similar to those in the lateral walls is situated ventral to the canal area. Preliminary data indicated that the cells in this cluster were extremely susceptible to ionizing radiation; the present study: (1) characterizes these cells ultrastructurally in the normal animal; and (2) examines radiation-induced changes. Ultrastructurally, the cells forming this cluster in the normal 3-day-old rat were tightly packed, and their outlines followed the contours of adjacent cells. The cytoplasm, which contained an abundance of organelles, including distinct, wide-bore endoplasmic reticulum with darkly stained cisternae, occasionally surrounded bundles of axons. Synapses and junctional complexes were absent. Both nuclear and cytoplasmic characteristics are those of immature astrocytes 9 . By 6 h following irradiation of the 3-day-old, many cells in this cluster were pyknotic, and by 24 h the cluster had essentially disappeared. The number of cells surrounding the lumen decreased less rapidly than in the cluster. By 10 days post-irradiation (P-I) this number was approximately 39% less than in the control. These changes indicated a differential sensitivity to ionizing radiation between the cells of the ventral cluster and those elsewhere in the central canal area.

This study examined, during normal development and during the development of a glial cell deficient axon population, the nature of astrocyte involvement at the central nodes of Ranvier on spinal cord axons. One condition examined was the ventral funiculus of normal 7-day-old rats. At this age, the lumbar spinal cord underwent an active phase of gliogenesis, and axons were seen in various stages of myelination. Perinodal astrocytic processes were routinely observed at nodes of axons on which myelin sheaths exceeded 8 compact lamellae. Perinodal astrocytic processes were also seen in close proximity to axolemma at most developing nodes. This study also examined the lumbar spinal cords of rats which were X-irradiated on the third postnatal day. This procedure caused a profound reduction in the astrocyte and oligodendrocyte population in 13- and 18-day-old rats, while sparing the neuronal elements. Thus, axo-glial relationships observed in this tissue are unlikely to be random occurrences. Despite the reduction in glial cells, some oligodendrocyte-myelinated axons were observed in the irradiated spinal cords. Perinodal astrocytes were seen at all oligodendrocyte-derived nodes observed in the irradiated cord and appeared to have a specific relationship to the node of Ranvier. The presence of astrocytic processes at the normal, developing node and at the nodes in glial cell deficient spinal cords suggests that astrocytes may be necessary to the function of nodal axolemma. In irradiated spinal cords, where the glial cells are markedly reduced, apposition between astrocytic and oligodendrocytic membrane at the paranode and internode was also seen and was so common that it is highly unlikely to be due to random occurrences. These observations further suggest that in addition to the presumptive role at the nodes, astrocytes may play an inductive or supportive role in the development and maintenance of central myelin.

Relationships between intraspinal Schwann cells and neuroglia, particularly, astrocytes, were studied following X-irradiation of the spinal cord in 3-day-old rats. Initially, this exposure results in a depletion of the neuroglial population. By 10 days post-irradiation (P-I), gaps occur in the glia limitans, although the overlying basal lamina remains intact. Development of and myelination by intraspinal Schwann cells is well underway by 15 days P-I. These Schwann cell-occupied regions have a paucity of astrocyte processes, a finding which persists throughout the study (60 days P-I), and several types of Schwann cell-neurologial interfaces are observed, including: (1) astrocyte separation of Schwann cells from oligodendrocyte-myelinated regions; (2) intermingling of Schwann cell-myelinated axons and oligodendrocyte-myelinated axons in the absence of astrocyte processes; and (3) ensheathment of unmyelinated axons by astrocyte processes which separate these axons from the Schwann cells. The gaps in the glia limitans widen as the P-I interval increases. At 45 and 60 days P-I, the basal lamina no longer forms a singular, continuous covering over the spinal cord surface, but follows instead a rather tortuous course over the disrupted glia limitans and the intrasponal Schwann cells. Although the mode of initial occurence of Schwann cells within the spinal cord is not yet understood, the data indicate that the astrocyte population is involved in that process, as well as in limiting the further development of Schwann cells within the substance of the spinal cord.

Schwann cells, peripheral-type myelin and connective tissue elements develop within the dorsal portion of the X-irradiated spinal cord in immature rats. Factors controlling the distribution of these elements within the irradiated site are not fully understood. In the present study [3H]thymidine autoradiography was used to examine proliferative activities of cells in these areas occupied by peripheral nervous system components, and correlative ultrastructural evaluations were made. At 15 and 20 days post-irradiation (P-I), the Schwann cells occupied the dorsolateral portions of the dorsal funiculi, and heavily labeled cells occurred throughout these areas. By 25 days P-I the Schwann cells extended ventrally into the depths of the dorsal funiculi and into the dorsal gray matter, and labeled cells were concentrated in the deeper portions of these areas. Ultrastructurally, the Schwann cells and peripheral-type myelin were more mature in the superficial portions where proliferative activity was diminished. In contrast, much less mature, peripheral-type myelin occurred in the depths where the labeled cells were concentrated. At 30 and 45 days P-I, labeled cells were much less frequent but usually occurred in the depths when observed. Similarly, a dorsal-ventral gradient in maturity of peripheral-type myelin was evident ultrastructurally. By 60 and 90 days P-I, labeling was rare, and mature Schwann cell myelin was present throughout the areas. Astrocytes and their processes were less numerous in regions invaded by Schwann cells, as compared to controls, and studies are in progress to evaluate the relationships between these glial elements and intraspinal peripheral nervous system components.

Following irradiation, the dorsal funiculus of the lumbosacral spinal cord in the rat undergoes the following sequence of events: (a) a marked reduction of the normal glial population, (b) an absence of oligodendrocyte myelin formation, (c) the invasion and proliferation of Schwann cells, and (d) the myelination of axons within the cord by Schwann cells. The present study demonstrates that, during the latter process, junctional complexes develop between these intraspinal Schwann cells and the axolemma. These complexes are present at sites of probable initial contact between the two membranes. As the Schwann cell process begins to wrap the axons, these junctional complexes are located between the inner spiraling process of the Schwann cell and the axon. With the advancement of myelin formation to the stage of 8 to 9 compact spirals, these contacts are rarely observed. Spinal cords from normal 8-day-old rats were examined in order to determine if such contacts occur during myelination by oligodendrocytes. Although they are more difficult to detect in the normal animal due to the abundance of glial processes, similar junctional complexes occur between oligodendrocyte processes and axons. These observations suggest that these complexes may serve to stabilize and to guide the myelin-forming process around the perimeter of the axon. Additionally, these junctions may play an active role in the advancement of the inner spiraling process by forming temporary adhesions between the axolemma and the adjacent myelin-forming process. Coated vesicles are commonly observed fused with the axolemma of axons which are in the early stages of myelination. These coated vesicles may be involved in the insertion or the deletion of junctional membrane.