Morphine does not affect astrocyte survival in developing primary mixed-glial cultures

In mixed-glial cultures, high concentrations of morphine (1 microM) have previously been shown to completely inhibit any increase in glial numbers, although DNA synthesis continues in flat, polyhedral astrocytes (type 1 astrocytes). This suggests that high concentrations of morphine are toxic to glia. Morphine toxicity was assessed in mixed-glial cultures using calcein-AM and ethidium homodimer dyes as viability markers to identify live and dead cells, respectively. At 3, 5, and 7 days in vitro there was no significant difference in the number of dead cells between untreated and opiate-treated groups. Comparable numbers of ethidium homodimer-labeled cells were present in all groups. The greatest amount of cell death (16-19%) occurred at 3 days in vitro, while fewer cells (8-12%) were dying at 7 days in vitro. To further characterize the dying glia, glial fibrillary acidic protein (GFAP) and A2B5 immunocytochemistry were combined with viability markers. Only GFAP immunoreactive process-bearing cells and A2B5 immunoreactive cells (process-bearing cells and possibly some neurons) were dying in culture, whereas the death of flat, polyhedral GFAP-positive cells was not observed. Cell survival was not affected by morphine, but may be affected by culture conditions. Thus, morphine-induced reductions in glial numbers did not result from an increased rate of cell death. Collectively, the present and previous findings suggest that morphine inhibits the production of flat, polyhedral astrocytes solely by decreasing their rate of proliferation.