Morphological, immunophenotypical and electrophysiological properties of resting microglia in vitro

Abstract

Morphological, immunophenotypical and electrophysiological properties were investigated in isolated cultured murine microglia before and after exposure to astrocyte-conditioned medium (ACM). Following application of ACM, microglial cells underwent a dramatic shape transformation from an amoeboid appearance to a ramified morphology. In parallel to morphological changes, a downregulation of macrophage surface antigens was observed in microglia exposed to ACM. Staining intensities for major histocompatibility complex (MHC) class II molecules and for the adhesion molecules leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) were significantly decreased in ramified microglia 5 days after exposure to ACM. In microglial cells treated daily with ACM over a period of 5 days, the smallest staining intensities for all surface antigens as well as the smallest ramification index as a measure for the highest degree of ramification were determined. In addition, upregulation of delayed rectifier K + currents was observed in microglia exposed to ACM for 1 day or treated daily with ACM for 5 days. In contrast, untreated amoeboid microglia or ramified microglia analysed 5 days after exposure to ACM did not express delayed rectifier K + currents. Analyses of the resting membrane potential and expression levels and properties of inward rectifier K + currents did not reveal any differences between untreated and ACM-treated microglia. It is suggested that electrophysiological properties of microglia do not strongly correlate with the morphology or the immunophenotype of microglial cells.

The tyrosine kinase inhibitor AG126 restores receptor signaling and blocks release functions in activated microglia (brain macrophages) by preventing a chronic rise in the intracellular calcium level

“…We first compared calcium signals to C5a application in microglial cells of standard cultures and in microglial cells with ramified morphology that were cultivated in two different conditioned media and resembled the 'resting' microglia in vivo (Eder et al 1999;Kloss et al 2001). Under all three culture conditions, most of the cells responded with marked calcium signals irrespective of their morphological appearance (Figs 2c-e).…”

Regulation of a TRPM7-like Current in Rat Brain Microglia

“…For electrophysiology, microglia were plated on 15-mm sterile glass coverslips and cultured in either serum-free medium (i.e. MEM with 2% B27 supplement (Invitrogen), 2 mM L-glutamine, and 0.05 mg/ml gentamycin) or astrocyte-conditioned medium (ACM), collected as the supernatant from rat astrocyte cultures grown in MEM with 2% fetal bovine serum (21). ACM was collected twice a week, frozen at Ϫ70°C, and used within one month.…”

“…An Outward-rectifying Cation Current-Unless otherwise indicated, all recordings were from microglia that were cultured in serum-free medium or astrocyte-conditioned medium, conditions that reduce microglia activation (21). We patch-clamped microglia that were unipolar or bipolar with elongated cell bodies, as in one of our earlier studies (12).…”

GM1 induces p38 and microtubule dependent ramification of rat primary microglia in vitro

“…Several studies showed that astrocytes play a significant role in the differentiation and transformation. Ameboid microglial cells have been shown to ramify when they are cultured on monolayers of astrocyte (Liu et al, 1994;Sievers et al, 1994;Tanaka and Maeda, 1996) or treated in vitro with astrocyteconditioned medium (Eder et al, 1999;Suzumura et al, 1990). Astrocyte-derived granulocyte/macrophage-colony stimulating factor and macrophage colony stimulating factor can induce microglial ramification in vitro (Ohsawa et al, 1997;Schilling et al, 2001;Suzumura et al, 1990), and astrocytes release serine and glycine into the culture medium and both amino acids enhance ramification of microglial cell (Tanaka et al, 1998).…”