Ballistic labeling and dynamic imaging of astrocytes in orcranotypic hippocampal slice cultures

Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchiteciture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive, processes, ascrocytes situated,in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Downs of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or calistically labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture. development and dynamic remodeling of astrocyte's and their relationships to neurons and glia in live mammalian brain tissues. (C) 2004 Elsevier B.V. All rights reserved.