We investigated the cellular mechanisms underlying the Ca(2+)-dependent release of glutamate from cultured astrocytes isolated from rat hippocampus. Using Ca(2+) imaging and electrophysiological techniques, we analyzed the effects of disrupting astrocytic vesicle proteins on the ability of astrocytes to release glutamate and to cause neuronal electrophysiological responses, i.e., a slow inward current (SIC) and/or an increase in the frequency of miniature synaptic currents. We found that the Ca(2+)-dependent glutamate release from astrocytes is not caused by the reverse operation of glutamate transporters, because the astrocyte-induced glutamate-mediated responses in neurons were affected neither by inhibitors of glutamate transporters (beta-threo-hydroxyaspartate, dihydrokainate, and L-trans-pyrrolidine-2,4-dicarboxylate) nor by replacement of extracellular sodium with lithium. We show that Ca(2+)-dependent glutamate release from astrocytes requires an electrochemical gradient necessary for glutamate uptake in vesicles, because bafilomycin A(1), a vacuolar-type H(+)-ATPase inhibitor, reduced glutamate release from astrocytes. Injection of astrocytes with the light chain of the neurotoxin Botulinum B that selectively cleaves the vesicle-associated SNARE protein synaptobrevin inhibited the astrocyte-induced glutamate response in neurons. Therefore, the Ca(2+)-dependent glutamate release from astrocytes is a SNARE protein-dependent process that requires the presence of functional vesicle-associated proteins, suggesting that astrocytes store glutamate in vesicles and that it is released through an exocytotic pathway.

Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle-or fusion-related mechanism.During the past decade there has been increasing evidence for both integrative and dynamic roles for astrocytes in the central nervous system. Following activation of G protein-coupled receptors, astrocytes exhibit calcium oscillations, leading to the release of the chemical transmitters glutamate and ATP (1-3). Studies in vitro and in brain slices have led to the hypothesis of tripartite synaptic transmission (4); neuronal activity causes elevations of synaptically associated calcium in astrocytes, which in turn leads to the release of chemical transmitters from these glial cells to locally modulate synaptic transmission (2, 5-7).The mechanisms mediating the release of these transmitters from astrocytes are, however, ill-defined and are still the subject of intense debate. At least three distinct release pathways have been proposed as mediating the calcium-dependent release of glutamate from astrocytes: the reversal of plasma membrane glutamate transporters, anion transporter mediate release mechanisms, and calcium-dependent exocytosis (8 -10). Because the release of glutamate is stimulated by calcium elevations and is not affected by glutamate transport inhibitors and because changes in cell volume have not been detected coincident with release, it has been proposed that this transmitter is released through a vesicle-mediated exocytotic pathway.Several observations made using cultured astrocytes support such a vesicle-mediated exocytotic mechanism of glutamate release, including the calcium depend...

Werner syndrome is an autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several lines of evidence have suggested that the Werner syndrome protein WRN plays a role in DNA replication and Sphase progression. In order to define the exact role of WRN in genomic replication we examined cell cycle kinetics during normal cell division and after methyl-methane-sulfonate (MMS) DNA damage or hydroxyurea (HU)-mediated replication arrest following acute depletion of WRN from human fibroblasts. Loss of WRN markedly extended the time cells needed to complete the cell cycle after either of these genotoxic treatments. Moreover, replication track analysis of individual, stretched DNA fibers showed that WRN depletion significantly reduced the speed at which replication forks elongated in vivo after MMS or HU treatment. These results establish the importance of WRN during genomic replication and indicate that WRN acts to facilitate fork progression after DNA damage or replication arrest. The data provide a mechanistic basis for a better understanding of WRN-mediated maintenance of genomic stability and for predicting the outcome of DNA-targeting chemotherapy in several adult cancers that silence WRN expression.

The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology-consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium-has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). Micro fabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, the medium composition, as well as the type of neighboring cells surrounding the microenvironment of the cell. In addition, microtechnology is conceptually well suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. Here we review a variety of applications of microfabrication in cell culture studies, with an emphasis on the biology of various cell types.

During embryonic neural development, axon tips ("growth cones") are guided through a dynamic three-dimensional (3-D) landscape by soluble chemotropic factors and by immobilized, growth-permissive or growth-inhibiting contact cues present in the extracellular matrix and on the surface of surrounding cells. It has been difficult to probe the search algorithms of growth cones in response to multiple contact cues during 3-D navigation using traditional two-dimensional (2-D) substrates. Here, we present an in vitro study in which the axons of murine embryonic cortical neurons are challenged with competing growth options, using 3-D substrates that feature variations in permissiveness and microtopography. As 3-D substrates, we used poly-D-lysine (PDL) coatings on microfabricated steps of polydimethylsiloxane (PDMS) and complementary features of Matrigel. We found that axons display a preference for PDL over Matrigel and for the straightest path within a distance consistent with the exploratory range of the growth cone. When these two preferences are in conflict, axons choose to grow straight into Matrigel; when the straight path is not permissive, the axon turns in the direction that minimizes the turning angle. These results suggest that growth cones make 3-D navigation decisions by integrating permissiveness and topographical cues.