Autocellular coupling by gap junctions in cultured astrocytes: a new view on cellular autoregulation during process formation.

Neocortical astrocytes make two types of gap junctions, intercellular ones create a functional syncytium, while reflexive gap junctions mediate autocellular coupling and serve unknown functions (Rohlmann and Wolff, 1996). Here, the question is addressed whether solitary astrocytes in vitro express connexin43 (Cx43) and establish gap junctions in the absence of intercellular contacts. In all media conditions tested, immunocytochemistry visualized Cx43-expression and gap junctions irrespective of the presence or absence of intercellular contacts. Reflexive gap junctions were associated with mechanical junctions (adherent spots and fascia adherens) connecting surface membranes and cytoskelal components, respectively. Both were characteristically located along incompletely separated borders between developing processes and/or branches. In addition, Cx43-immunoreactivity was found on some non-junctional membranes: i) intracellular vesicle clusters sited to forming processes and at the basis of filopodia; ii) the surface membrane of filopodial subpopulations usually appearing in bunches. Results suggest changes in the resumptive role of Cx43 in cultivated astrocytes: 1) Cx43 is not confined to intercellular gap junctions, it may even selectively compose reflexive ones; 2) from intracellular stores (vesicle aggregates), Cx43 may be incorporated into the surface membrane of filopodia; 3) by contacting other parts of the same cell surface (or neighboring cells), filopodia and membrane patches carrying Cx43-half channels may be essential in initial steps of gap junction formation; 4) the distribution of reflexive gap junctions is compatible with the hypothesis that autocellular coupling serves reorganization of cytoskeleton during the formation of cell processes and branches; 5) in general, gap junctions may be important for coordinating the cytoskeleton across intercellular contacts and within cells with complex shape.

Lack of excitotoxic cell death in serum-free cultures of rat cerebral cortex.

The excitotoxicity of glutamate, N-methyl-D-aspartate (NMDA), quisqualate and kainate were compared in rat cerebro-cortical cell cultures grown in different media. Excitotoxic cell death was examined by phase contrast microscopy and by the measurement of lactic dehydrogenase (LDH) activity released in the culture medium. Cells grown for 14 days in a serum-free, defined (N2) medium were resistant, while sister cultures maintained in the presence of serum responded to the excitotoxins by a significant degree of cell death. Cells cultured in serum-free medium for 7 days, and fed by a serum-containing one for an additional week exhibited vulnerability to the excitotoxins. It is concluded that the development of excitotoxicity in rat cortical cultures depends on one or more serum constituents.

Interaction of soluble and substratum-bound factors influences neurite elongation.

Neurite elongation was investigated in explanted chick dorsal root ganglia (DRG) using either serum-supplemented or defined N1 medium and different culture substrata. The results indicate that there is no substantial difference in neurite elongation on the three substrata when ganglia are grown in N1 medium. In contrast, neurite length depends on the substratum in serum-supplemented medium and in general exceeds that obtained in N1 medium. Optimal neurite growth was observed in N1 medium when culture dishes have been precoated with serum. Our data suggest that while soluble serum molecules inhibit neurite growth adsorbed serum constituents clearly stimulate neurite elongation.