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While the astrocytic control of extracellular glutamate concentration at synaptic contacts is well characterized, little is known regarding the clearance of glutamate along axon tracts, even though local excitotoxic damage has been reported. Therefore, we have compared glutamate handling in astrocyte cultures derived from white matter (corpus callosum) and grey matter tissues (cortical structures). These populations of astrocytes showed clearly distinct phenotypes, adopting stellate or protoplasmic morphologies respectively. In addition, white matter astrocytes showed high densities of the intermediate filament proteins glial fibrillary acidic protein, vimentin and nestin. The glutamate-aspartate transporter and glutamate transporter-1, as well as glutamine synthetase, were found to be expressed at higher levels in white matter compared with grey matter astrocytes. Consistent with this aspartate uptake capacity was three to fourfold higher in white matter cells, and the use of specific inhibitors revealed a substantial activity of glutamate transporter-1, contrasting with grey matter cells where this transporter appeared poorly functional. In addition, expression of type 5 metabotropic glutamate receptors was considerably higher in white matter astrocytes where the agonist (S)-3,5-dihydroxyphenylglycine triggered a large release of intracellular calcium. Differences in these astrocyte cultures were also observed when exposed to experimental conditions that trigger glial activation. This study highlights typical features of cultured astrocytes derived from white matter tissues, which appear constitutively adapted to handle excitotoxic insults. Moreover, the expression and activity of the astroglial components involved in the control of glutamatergic transmission are reinforced when these cells are maintained under conditions mimicking a gliotic environment.

S100A4 is a member of the EF-hand family of calcium-binding proteins, first identified in tumor cells, and implicated in tumor invasion and metastasis. Intracellular upregulation of S100A4 is associated with increased motility of tumor cells. Extracellular application of S100A4 increases the motility of glioma cells in vitro. We showed previously that astrocytes in spinal cord and brain white matter also express S100A4. This expression is markedly increased in reactive white matter astrocytes after injury. Here, we have explored how changes in intracellular S100A4 affect migration of astrocytes. We produced cultures of white matter, S100A4 expressing astrocytes, and developed a small interfering (si) RNA approach to specifically eliminate S100A4 expression in these cells, and compared the migration of astrocytes expressing S100A4 with astrocytes transfected with S100A4 siRNA. As a "positive control" we used S100A4 expressing C6 glioma cells. In contrast to malignant cells, S100A4 expressing astrocytes increased their migration capacity after S100A4 siRNA treatment. At the same time, and in parallel with increased migration, white matter astrocytes increased their expression of metalloproteinases MMP-9 and MT1-MMP. The addition of MMP-2/MMP-9 inhibitor resulted in a significant inhibition of migration in S100A4 siRNA-treated astrocytes. These findings indicate that S100A4 has a stabilizing function in reactive white matter astrocytes, a function that may contribute to the development of a rigid, growth-inhibitory glial scar., ((c) 2005 Wiley-Liss, Inc.)

White matter astrocytes have physiological functions which are distinct from those of astrocytes in gray matter. White matter becomes highly non-permissive to neurite growth after injury, but the role of white matter astrocytes in this process is incompletely understood. Current protocols for making primary astroglial cultures are inadequate for exploring the specific properties of white matter astrocytes in vitro. We describe a procedure for obtaining cultures of white matter astrocytes from the rodent corpus callosum. In this procedure, we take advantage of our previous finding that white, but not gray matter astrocytes express the calcium-binding protein S100A4. S100A4 expressing astrocytes are abundant in the corpus callosum, and we show that cultures, highly enriched in S100A4 expressing white matter astrocytes, can be reproducibly generated from this area. Key factors for successful cultures are (i) meticulous dissection of the corpus callosum from 4-day-old rats, and (ii) Percoll density gradient centrifugation to purify astrocytes. As a means of exploring the possible role of S100A4 in white matter astrocytes, we describe the use of the siRNA technique to eliminate the expression of S100A4 in our in vitro system.

Nerve fibre degeneration in the spinal cord is accompanied by astroglial proliferation. It is not known whether these cells proliferate in situ or are recruited from specific regions harbouring astroglial precursors. We found cells expressing nestin, characteristic of astroglial precursors, at the dorsal surface of the spinal cord on the operated side from 30 h after dorsal root injury. Nestin-expressing cells dispersed to deeper areas of the dorsal funiculus and dorsal horn on the operated side during the first few days after injury. Injection of bromodeoxyuridine (BrdU) 2 h before the end of the experiment, at 30 h after injury, revealed numerous BrdU-labelled, nestin-positive cells in the dorsal superficial region. In animals surviving 20 h after BrdU injection at 28 h postlesion, cells double-labelled with BrdU and nestin were also found in deeper areas. Labeling with BrdU 2 h before perfusion showed proliferation of microglia and radial astrocytes in the ventral and lateral funiculi on both sides of the spinal cord 30 h after injury. Nestin-positive cells coexpressed the calcium-binding protein Mts1, a marker for white matter astrocytes, in the dorsal funiculus, and were positive for glial fibrillary acidic protein (GFAP), but negative for Mts1 in the dorsal horn. One week after injury the level of nestin expression decreased and was undetectable after 3 months. Taken together, our data indicate that after dorsal root injury newly formed astrocytes in the degenerating white and grey matter first appear at the dorsal surface of the spinal cord from where some of them subsequently migrate ventrally, and differentiate into white- or grey-matter astrocytes.

We recently showed that Mts1 is expressed in white matter astrocytes in the rat brain and spinal cord from the first postnatal day. Its expression level declined in the adult CNS, but its topographical localization was maintained. Only white matter astrocytes in the cerebellum did not express Mts1. After dorsal root or sciatic nerve injury, we observed a marked upregulation of Mts1 in the area of the dorsal funiculus undergoing Wallerian degeneration. Here we show that upregulation of Mts1 is a consistent feature of astrocytes in white matter undergoing Wallerian degeneration. In addition, Mts1 is upregulated in astrocytes outlining the lesion site of a penetrating injury to the forebrain, or cerebellum. Gray matter astrocytes did not express Mts1, even after direct injury. In injured brain, we consistently noted a close relationship between Mts1-expressing astrocytes and ED1-positive microglia/macrophages, which are known to be highly motile cells. Mts1 was expressed in the periventricular area and the rostral migratory stream, i.e., sites of ongoing neuroplasticity in adulthood, and was upregulated in these areas after injury. These data suggest that Mts1-expressing astrocytes play a significant role in degenerative events in the mature white matter, interact with phagocytic microglia/macrophages and regulate cell migration and differentiation in areas of the adult brain with a high degree of plasticity., (Copyright 2002 Wiley-Liss, Inc.)