title: Induction of resting microglia in culture medium devoid of glycine and serine.
abstract: Cultured microglial cells usually exhibit ameboid morphology and peripheral macrophage-like properties, which are distinct from those observed in the normal mature brain. This might be caused by the inappropriate culture of microglial cells in high concentrations (approximately 200-400 microM) of Gly and Ser, although the concentrations of the amino acids in extracellular spaces of the brain parenchyma are quite low (approximately 5 microM). In the present study, we focused on the concentration-dependent effects of glycine (Gly) and serine (Ser) on microglial morphology and function. Under Gly/Ser-free and serum-free condition, the majority of rat microglial cells displayed round morphology, whereas in the presence of 5 microM Gly and 25 microM Ser, which correspond to the concentrations of Gly and Ser in the cerebrospinal fluid, they extended multiple branched processes and formed clusters of rough endoplasmic reticulum. On the other hand, Gly and Ser did not affect morphology of astrocytes. The viability of microglia was not affected by the changes in the concentrations of Gly and Ser. Metabolic activity, activities of acid phosphatase and inducible nitric oxide synthase, and superoxide anion (O2-) generation were all strongly suppressed in Gly/Ser-free medium or in medium containing physiological concentrations of both amino acids. Such activities were all enhanced in harmony with increases in the concentrations of Gly and Ser. Thus, microglial cells cultured in Gly/Ser-free medium, even though exhibiting ameboid morphology, appears to be in the functionally resting state. Furthermore, once the resting state was achieved, the microglial cells remained inactive even after the subsequent 24 h culture in serum-supplemented medium containing 400 microM of both amino acids. The medium conditioned by microglial cells that were cultured in the presence of 400 microM of Gly and Ser was toxic to cortical neurons, whereas the microglia-conditioned medium obtained in the absence of both amino acids facilitated the survival of cortical neurons. Therefore, microglial cells in the resting state, which was induced in the Gly/Ser-free condition, are likely to support neurons. Microglial cells could ramify on glass coverslips coated with astrocyte-derived extracellular matrix or on coverslips coated thinly with fibronectin and/or laminin even under the Gly/Ser-free condition. The ramified cells as induced in this way kept suppressed O2- 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.
authors.affiliation: Department of Physiology, School of Medicine, Ehime University, Japan. jtanaka@m.ehime-u.ac.jp
authors.forename: J
authors.initials: J
authors.lastname: Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Toku
authors.forename: S
authors.initials: S
authors.lastname: Matsuda
authors.forename: S
authors.initials: S
authors.lastname: Sudo
authors.forename: H
authors.initials: H
authors.lastname: Fujita
authors.forename: M
authors.initials: M
authors.lastname: Sakanaka
authors.forename: N
authors.initials: N
authors.lastname: Maeda
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004396:Coloring Agents; D003470:Culture Media; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis; D005998:Glycine / Q000502:physiology*; D007797:Laminin / Q000096:biosynthesis; D008262:Macrophage Activation / Q000502:physiology*; D017628:Microglia / Q000502:physiology*; D008854:Microscopy, Electron; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D010078:Oxazines; D010101:Oxygen Consumption / Q000502:physiology; D051381:Rats; D012694:Serine / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D014343:Trypan Blue; D014966:Xanthenes
chemical_list: D004396:Coloring Agents; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D007797:Laminin; D010078:Oxazines; D014966:Xanthenes; D013481:Superoxides; C005843:resazurin; D012694:Serine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D000135:Acid Phosphatase; D014343:Trypan Blue; D005998:Glycine