Astrocytes and their processes play a role in guiding the migration and axonal growth of neurons during embryonic development 1,2) or in establishing the blood-brain barrier. [3][4][5] Furthermore, astrocytes become reactive in response to brain tissue damage caused by hypoxia, ischemia or seizures.
6,7)Reactive astrocytes are characterized by stellate morphology and an increased expression of glial fibrillary acidic protein.Therefore, the study of mechanisms regulating astrocyte morphology is very important for understanding brain development and brain response to injury or disease. Cultured astrocytes from the cerebral cortex of neonatal rats display flattened, polygonal morphology in the absence of stimuli, and change into process-bearing stellate cells in response to specific stimuli, e.g., cyclic AMP analogs, [8][9][10] phorbol ester [11][12][13] or Alzheimer's disease amyloid b protein.14-17) This morphological change is termed stellation and has been widely studied for understanding the regulatory mechanisms of astrocyte morphology.L-Glutamate functions as a major excitatory neurotransmitter in the central nervous system. Astrocytes possess an abundance of molecules that could be the target of L-glutamate, e.g., glutamate receptors or glutamate transporters. 18,19) However, the functional roles of L-glutamate in astrocytes are not fully understood. We have recently found that L-glutamate suppresses the astrocyte stellation induced by amyloid b protein, cyclic AMP analog or phorbol ester in culture, suggesting that L-glutamate is a regulator of astrocyte morphology.17) The suppressive effect of L-glutamate on astrocyte stellation was not mimicked by glutamate receptor agonists, and not blocked by glutamate receptor antagonists. In contrast, the effect of L-glutamate was mimicked by D-and L-aspartate and transportable glutamate uptake inhibitors. We hypothesized that L-glutamate suppresses astrocyte stellation through a mechanism related to the glutamate transporter. To support this idea, we investigated the Na ϩ and K ϩ dependence of this effect of L-glutamate in the present study. The glutamate transporter is driven by Na ϩ influx and K ϩ efflux, 20) and the plasma membrane Na ϩ -K ϩ pump serves to maintain the gradient of extracellular and intracellular Na ϩ or K ϩ concentrations. If the effect of L-glutamate on astrocyte morphology requires the activity of glutamate transporters, it should be attenuated by lowering Na ϩ and K ϩ gradients or by inhibiting the Na
MATERIALS AND METHODSPrimary cultures of astrocytes were prepared from the cerebral cortices of 2-d-old neonates of Wistar rats, as described previously.21) Briefly, dissociated cortical cells were suspended in modified Eagle's medium containing 30 mM glucose, 2 mM glutamine, 1 mM pyruvate and 10% fetal bovine serum, and plated on uncoated 25 cm 2 flasks at a density of 600000 cells/cm 2 . The monolayer of type I astrocytes was obtained 12-14 d after the plating. Non-astrocytes such as microglia were detached from the flasks by shaking, then remo...