Specificity of cell-cell coupling in rat optic nerve astrocytes in vitro.

Abstract

Intercellular coupling was studied in cultured rat optic nerve astrocytes individually characterized by A2B5 antibody staining. The presence ofcell coupling was assessed by injecting single cells with the low molecular weight fluorescent dye Lucifer yellow and noting dye passage into adjacent cells; cell coupling was also studied by analyzing the decay phase of current transients recorded in response to small voltage steps using whole-cell patch-clamp recording. Cell coupling was restricted to A2B5-astrocytes, the majority of which had a flat fibroblast-like appearance and was never observed in A2B5+ stellate-shaped astrocytes. Furthermore, A2B5-astrocytes showed coupling only to A2B5-and never to A2B5' astrocytes.Analysis of current transients provided an additional indicator for cell coupling. Astrocytes that showed dye coupling to at least one neighboring cell required the sum of two exponential functions to fit current transients, whereas a single exponential function sufficed to fit transients in cells that were not dye coupled. The specificity of cell coupling in cultured rat optic nerve astrocytes suggests that predominantly A2B5-astrocytes comprise a coupled glial syncytium; this physiological feature of these cells may be a specialized adaptation for "spatial buffering," the transport of K+ away from areas of focal extracellular accumulation. On the other hand, A2B5M astrocytes form an uncoupled subpopulation of rat optic nerve glial cells that may serve different functions.Astrocytes form gap junctions with adjacent astrocytes (1) and these large intercellular channels are believed to link the cells into a three-dimensional network. As previously shown for a variety of cell systems, these connections allow direct cytoplasmic diffusion of inorganic and small organic molecules between cells (2). Injection of the low molecular weight fluorescent dye Lucifer yellow (LY) (3) into single astrocytes has revealed dye spread into adjacent astrocytes, thereby indicating gap junctional communication, in cortical slices (4), spinal cord (5), cortical (6) and cerebellar cell cultures (7), and rat optic nerve (RON) in situ (8). Similarly, coupling of cultured astrocytes was apparent in studies in which laser photobleaching techniques were used (9).Cultures from RON provide a well-described glial cell system in which two types of astrocytes can be distinguished morphologically and by their ability to bind the monoclonal antibody A2B5 (reviewed in ref. 10). In vitro A2B5-/GFAP+ (glial fibrillary acidic protein) astrocytes [termed type 1 astrocytes, (11)] have a fibroblast-like appearance and are believed to develop from a specific, still poorly characterized, progenitor cell. In contrast, A2B5+/GFAP+ astrocytes [termed type 2 astrocytes (11)] have a stellate appearance and share a common precursor cell, the 02A progenitor cell, with oligodendrocytes (10,11). Further differences between these types of astrocytes have become apparent and include, but are not limited to, their response to and the secretion of gro...

Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation

“…A similar developmental pattern was described for junctional communication among astrocytes in rat visual cortex (Binmoller and Muller, 1992) and rat somatosensory cortex (Connors et al, 1983). In cultures of cerebellum and optic nerve, immature astrocytes are uncoupled; with maturation, astrocytes form large syncytia (Fischer and Kettenmann, 1985;Sontheimer et al, 1990). The gap junctional protein expressed by astrocytes is connexin 43 (Dermietzel et al, 1989;Yamamot0 et al, 1990; for review see Dermietzel and Spray, 1993).…”

Gene expression profiling of astrocytes from hyperammonemic mice reveals altered pathways for water and potassium homeostasis in vivo

“…Through this mechanism, astrocytes can facilitate spatial buffering of K + and spatial allocation of neurotransmitters and inter-astrocyte signalling over greater distances (Giaume et al, 1997;Hansson et al, 2000;Sontheimer et al, 1990;Wallraff et al, 2006). Therefore, the changes in Cx43, Kir channels and Aqp4 gene expression reported in this study are likely to have a significant impact on neuronal function and network activity during and after HA in the brain.…”

“…A very important function of the astrocyte syncytium and its gap junction channels is the maintenance of potassium homeostasis in brain (Sontheimer et al, 1990). The downregulation of Cx43 observed in FACS-purified GFAP-EGFP + astrocytes was analyzed further, because of: (i) the fundamental physiological role of Cx43 as the main gap junction channel in astrocytes (Dermietzel et al, 1991;Iacobas et al, 2004); (ii) the downregulation of Cx43 also found in cortical tissue of hyperammonemic mice (present study); (iii) the clustering and apparent loss of connectivity between EGFP + astrocytes along the brain vasculature of hyperammonemic animals (present study); and (iv) the importance of Cx43 for potassium homeostasis in the brain (Kozoriz et al, 2006;Wallraff et al, 2006).…”

Astrocytic Gap Junctions Remain Open during Ischemic Conditions

“…Characteristically, very large currents were obtained by varying the applied voltage during resting conditions (Fig. 4 A), as would be expected from cells that are coupled extensively (Sontheimer et al, 1990;Bordey and Sontheimer, 1997). For example, a 60 mV hyperpolarization activated a current of Ϫ1.23 Ϯ 0.16 nA (n ϭ 7).…”

“…Whole-cell current recorded from coupled astrocytes is a function of both their gap junction conductance and conductance through other membrane channels, in particular K-channels (Sontheimer et al, 1990;Sontheimer and Waxman, 1993). To establish the relative contribution of gap junctions to whole-cell current, we next patch-clamped astrocytes from the slice surface (Fig.…”