INDUCTION OF RESTING MICROGLIA IN CULTURE-MEDIUM DEVOID OF GLYCINE AND SERINE

Cultured microglial cells usually exhibit ameboid morphology and perip heral macrophage-like properties, which are distinct from those observ ed in the normal mature brain. This might be caused by the inappropria te culture of microglial. cells in high concentrations (similar to 200 -400 mu M) of Gly and Ser, although the concentrations of the amino ac ids in extracellular spaces of the brain parenchyma are quite low (sim ilar to 5 mu M). In the present study, we focused on the concentration -dependent effects of glycine(Gly) and serine (Ser) on microglial morp hology and function. Under Gly/Ser-free and serum-free condition, the majority of rat microglial cells displayed round morphology, whereas i n the presence of 5 mu M Gly and 25 mu M Ser, which correspond to the concentrations of Gly and Ser in the cerebrospinal fluid, they extende d multiple branched processes and formed clusters of rough endoplasmic reticulum. On the other hand, Gly and Ser did not affect morphology o f astrocytes. The viability of microglia was not affected by the chang es in the concentrations of Gly and Ser. Metabolic activity activities of acid phosphatase and inducible nitric oxide synthase, and superoxi de anion (O-2(-)) generation were all strongly suppressed in Gly/Ser-f ree medium or in medium containing physiological concentrations of bot h amino acids. Such activities were all enhanced in harmony with incre ases in the concentrations of Gly and Ser. Thus, microglial cells cult ured in Gly/Ser-free medium, even though exhibiting ameboid morphology , appears to be in the functionally resting state. Furthermore, once t he resting state was achieved, the microglial cells remained inactive even after the subsequent 24 h culture in serum-supplemented medium co ntaining 400 mu M of both amino acids. The medium conditioned by micro glial cells that were cultured in the presence of 400 mu M of Gly and Ser was toxic to cortical neurons, whereas the microglia-conditioned m edium obtained in the absence of both amino acids facilitated the surv ival of cortical neurons. Therefore, microglial cells in the resting s tate, which was induced in the Gly/Ser-free condition, are Likely to s upport neurons. Microglial cells could ramify on glass coverslips coat ed with astrocyte-derived extracellular matrix or on coverslips coated thinly with fibronectin and/or laminin even under the Gly/Ser-free co ndition. The ramified cells as induced in this way kept suppressed O-2 (-) generating activity. These findings suggest that resting ramified microglial cells with a neurotrophic activity can be induced with the combination of Gly/Ser-free medium and small amounts of extracellular matrix proteins, and that the resting state is rather stable. (C) 1998 Wiley-Liss, Inc.