N-methyl-D-aspartate Evokes Rapid Net Depolymerization of Filamentous Actin in Cultured Rat Cerebellar Granule Cells

Filamentous actin (F-actin) was measured in cultured rat cerebellum granule neurons with the use of fluorescently labeled phallotoxin as a site-specific probe for F-actin, and fluorescence microscopy. The averaged apparent intensity of soma-associated F-actin-derived fluorescence (F(app)) was measured from fixed cells after incubation in either 1) normal Krebs solution containing 2 mM extracellular calcium ([Ca2+]ex) or 2) normal Krebs solution plus N-methyl-D-aspartate (NMDA) for 2 min immediately before fixation. NMDA (10, 50, and 100 microM) decreased F(app) to 63 +/- 5% (mean +/- SE), 53 +/- 4%, and 47 +/- 2%, respectively, of that measured from control cells. This effect was mimicked by treatment of cells with ionomycin. The ability of NMDA to reduce the F(app) in the presence of [Ca2+]ex was abolished when cells were maintained in [Ca2+]ex-free medium. Cells first treated with NMDA for 2 min and then left in normal medium for 30 min before fixation gave F(app) fluorescence similar to control values (91 +/- 12%). However, if the F-actin polymerization inhibitor cytochalasin D was added to cells immediately after NMDA was removed, the F(app) did not recover with time (36 +/- 3%). Cells treated for 30 min with cytochalasin D alone showed a small reduction in staining (approximately 20%). It is concluded that the actin polymerization state of rat cerebellar granule neurons is sensitive to changes in intracellular calcium, and that NMDA receptor activation evokes an initial rapid depolymerization of F-actin.