Relationship between Na⁺ current expression and cell-cell coupling in astrocytes cultured from rat hippocampus

Cell-cell coupling between hippocampal astrocytes in culture was studied by following the intracellular spread of the low molecular weight fluorescent dye Lucifer yellow (LY). Dye coupling appeared as early as 24 h after plating, at which time ~20% of all astrocytes that physically contacted neighboring cells showed dye coupling. The percentage of coupled cells increased with time in culture and peaked after 10 days in vitro (DIV) when ~50% of astrocytes showed coupling. Further time in culture, up to 20 DIV, did not increase the percentage of coupled cells. Thus, coupled and noncoupled astrocytes coexist in hippocampal cultures in approximately equal numbers. Na⁺ currents were expressed in a subpopulation of hippocampal astrocytes and changed characteristics during in vitro development. A 'neuronal type' of Na⁺ current, so called because of an h(∞) curve that had a midpoint near -60 mV, was observed within the first 5 days post-plating. A 'glial type' of Na⁺ current, characterized by a -25 mV shift in its h(∞) curve, was only expressed after 6 days in culture. Na⁺ current expression was restricted to hippocampal astrocytes that did not exhibit dye coupling; astrocytes that exhibited dye coupling (n = 39) did not show measurable Na⁺ currents. The failure to see Na⁺ currents in coupled astrocytes cannot be explained by insufficient space-clamp since astrocytes acutely uncoupled with octanol (10 μM) did not reveal Na⁺ current expression. Control experiments showed that low concentrations of octanol (i.e., 10-100 μM) did not block Na⁺ currents; blockage of Na⁺ currents by octanol was only observed at high concentrations (e.g., 50-fold the concentration used for uncoupling). These observations support the idea that Na⁺-channel expression was restricted to noncoupled astrocytes. The time courses for the development of cell coupling and Na⁺ current expression appeared to be inversely correlated and suggested a gradual increase in cell coupling in concert with a loss in Na⁺ current expression with time in culture.