Stimulation of oligodendrocytes by extracts from astrocyte-enriched cultures

A soluble, heat-labile, trypsin-sensitive factor present in extracts of astrocyte-enriched cultures enhances the expression of myelination-associated events in rat brain cultures enriched in oligodendrocytes. Both the number of oligodendrocytes detected by immunofluorescence microscopy and the levels of biochemical activities associated with oligodendrocyte differentiation increased in the cultures in response to the extract in a time- and concentration-dependent fashion. The possible significance of these studies for normal oligodendrocytes function, and thus myelination, is discussed.     

Astrocyte cultures from adult rat brain. Derivation, characterization and neurotrophic properties of pure astroglial cells from corpus callosum

It has not as yet been routinely possible to derive primary cultures of glial cells from adult rat brain tissue even when adopting strategies that have proven successful with perinatal tissue. We now report that in response to a surgical lesion and a period of postoperative ‘priming’ in vivo, proliferating cultures of astroglial cells can be derived from the normally quiescent glia of the corpus callosum region of the adult rat brain. In such cultures the predominance of astroglia and the virtual absence of oligodendroglia and neurons has been established by the use of a variety of cell-type specific antisera. Fibroblasts, the only other cell type identified, when not numerous could be succesfully eliminated by treatment of the cultures with anti-Thy-1 antibodies and guinea pig compliment. Pure astroglial cells from adult brain have been sub-cultured and maintained for up to 4 months in vitro, providing suitable quantities of cells for studies on the trophic interaction between glia and neurons. In long-term culture the adult astrocytes maintain a flattened undiffirentiated morphology but readily assume a stellate shape with long branching processes upon the addition of a crude homogenate from bovine pituitary.     

In vitro studies on the comparative sensitivities of cells of the central nervous system to diphtheria toxin

We have used tissue culture techniques and cell type-specific antibodies to compare the sensitivity of the various cell types of the white matter tracts of the rat central nervous system to in vitro exposure to diphtheria toxin (DTx). We have found that oligodendrocytes and Type 2 astrocytes (Raff et al. 1983a), which at least in the rat optic nerve, appear to be derived from a single bipotential progenitor cell (Raff et al. 1983b), are both more susceptible to DTx than are either Type 1 astrocytes or spinal neurones. The loss of oligodendrocytes and Type 2 astrocytes caused by exposure to DTx in vitro appeared to be irreversible. Even when cultures were maintained for a month following initial treatment with DTx, these glial populations were not reestablished, suggesting that precursors for these macroglial cell types were as sensitive to the effects of DTx as were the oligodendrocytes and Type 2 astrocytes themselves. Our results are discussed in the light of the failure of diphtheritic lesions to remyelinate in vivo.     

Astrocytes in cell culture incorporate GM1 ganglioside

Ganglioside GM1 3H-labelled at the terminal galactose was added to astrocyte cell cultures. GM1 incorporation was studied in the two typical forms of astrocytes in cell culture of flat and stellate morphology. There was a strong time- and concentration-dependent increase in GM1 incorporation for both cell types of astrocytes. The incorporation of GM1 into the stellate form increased continuously up to 48 h (maximum time studied), while the incorporation into the flat form reached a plateau at the same time. After 2 h of GM1 incubation additional gangliosides appeared; the latter resulted from the metabolism of the GM1 incorporated, indicating that astrocytes in cell culture can biosynthesize more complex gangliosides. To confirm that GM1 was indeed incorporated into astrocytes, two other different approaches were used. Astrocyte cells treated with 3H-GM1 were visualized using autoradiography. The specific marker for GM1, rhodamine-labelled choleratoxin, was used to detect the incorporated GM1 using fluorescence microsocpy. In both cases GM1 treated cells were intensely labelled. These observations indicate that exogenous GM1 ganglioside can also be integrated into the astrocyte membranes as occurs in other types of cells and membranes.     

In vitro screening for anticonvulsant-induced teratogenesis in neural primary cultures and cell lines

To establish inherent potential for the induction of neural tube defects the ability of selected anticonvulsant agents to interfere with cell division has been established in vitro using an antiproliferative assay in clonal cell lines and a cytotoxicity assay using primary cultures of cerebral cortex neurons at different stages of development. In order to evaluate the relative toxicities of these agents their in vitro effects were determined at 2–3 times the plasma therapeutic level. By these procedures valproate and the benzodiazepines, diazepam and clonazepam, exerted a potent antiproliferative action which could not be attributed to increased cytotoxicity. In contrast phenytoin was markedly cytotoxic but was without an antiproliferative action. This cytotoxicity was most pronounced during the periods of extensive fibre outgrowth. When compared to epidemiological and animal study data, agents which inhibited cell proliferation within twice therapeutic concentration were consistently associated with major neural tube malformations. However phenytoin, found to be positive in the cell cytotoxicity assay, is not associated with neural tube malformations but rather is primarily associated with mental retardation. Thus assessment of antiproliferative activity of anticonvulsant drugs may be one criterion for identification of teratogenic potential during neurulation.     

Growth-associated protein 43 is down-regulated in cultured astrocytes

Growth-associated protein 43 (GAP-43; other designations—pp46, F1, B-50, p-57) is an abundant, neural, membrane-associated protein involved in synaptic plasticity and regeneration. We recently reported that GAP-43 is present in plasma membranes of cultured rat astrocytes. In the present study the level of astrocytic GAP-43 was assessed by indirect immunofluorescence labeling of cells with a specific anti-GAP-43 serum and immunoblotting of plasma membrane proteins. The results indicate that all astrocytes from 1-day-old cortex contained GAP-43 and those differentiated in culture did not. Furthermore, GAP-43 decreased dramatically during 3 weeks in culture. These results are consistent with the developmental down-regulation of GAP-43in vivo.     

Pathological reaction of astrocytes in perinatal brain injury

Summary Astrocytic reaction to various types of pre-and perinatal damage in the brain was studied using the immunohistochemical method for glial fibrillary acidic protein. The reactive gliosis could be detected as early as 20 weeks gestation. Reactive proliferation of the astrocytes could be seen already at 4 days after the insult. In addition to reacting to focal lesions, the astrocytes also proliferated diffusely throughout the white matter. The diffuse proliferation is the most significant finding in the evaluation of the perinatal damage, in both the acute state and in the long-term survivors.Supported in part by NIH Grant NS 06239. Part of the study was carried out while Dr. Roessmann was Visiting Professor at the University of Göttingen, supported by Deutsche Forschungsgemeinschaft, DFG-AZ: GO 76/100-1Presented in part at the IX International Congress of Neuropathology, Vienna, 1982     

Instruction-related changes of neuronal activity in area 5 during a simple forearm movement in the monkey

Unit recordings were made in area 5 of monkeys during the performance of a sound-triggered movement of the forearm. Changes in neuronal activity prior to the movement were observed in 188 neurons recorded in both normal and deafferented animals. When the discharge of these cells was analyzed as peristimulus histograms, it was seen that 152 neurons presented a pattern of discharge which was characterized by a brief modification in activity with a relatively constant latency after the auditory cue. Similar changes were observed in normal and deafferented animals but the latency was not the same for the two groups. These neurons may reflect the presence of a sensorimotor interface for the integration of instructions for movement and the subsequent genesis of motor commands.     

Effect of hyaluronidase on brain extracellular matrix in vivo and optic nerve regeneration

In the rat, intracerebral injection of bacterial hyaluronidase resulted in the almost complete disappearance of hyaluronic acid (HA) and glial hyaluronate-binding protein (GHAP) from cerebral hemispheres, brain stem, and cerebellum (but not from optic nerves and chiasm) starting 2–3 hr after the injection. HA and GHAP reappeared throughout the brain in characteristic patches 2–3 days after the injection. The patches gradually became confluent and after 12 days the brain appeared virtually normal. In normal rat optic nerve, staining for HA and GHAP ceased abruptly in the region of the lamina cribrosa. The retina was completely negative. HA and GHAP disappeared from hyaluronidase-injected optic nerve, chiasm, and contralateral optic nerve. In hyaluronidase-injected crushed optic nerves, regenerated axons were able to grow for short distances (about 500μm) into the distal stump undergoing Wallerian degeneration. No such growth was observed in saline-injected controls. © 1993 Wiley-Liss, Inc.     

Subependymal cells provide a faster response to ependymal injury than astrocytes in the hydrocephalic brain

Obstructive hydrocephalus was induced in 12-day-old rats by the infusion of kaolin into the cisterna magna. After 7 days, cortical and ependymal lesions were made with thin wire in non-hydrocephalic brains and in hydrocephalic brains. The wounds were allowed to heal for times ranging from 1 day to 10 days post-lesion, after which the animals were perfused and the brains prepared for histology. Paraffin sections of brain containing the ependymal wound were reacted for glial fibrillary acidic protein by the avidin biotin complex technique. Subependymal cells were associated with the ependymal defect from 1 day in both non-hydrocephalic and in hydrocephalic brains. Reactive astrocytes were identified in hydrocephalic wounded brains on day 3. It was not until day 6 that astrocytes and astrocytic processes were seen in association with the subependymal cells at the wound edge, and astrocytic processes were noted along the needle track in the cortex. The results suggest that the extremely rapid response of subependymal cells to ependymal injury is a mechanism distinct from the astrocytes response for repair in the brain.