MORPHINE DOES NOT AFFECT ASTROCYTE SURVIVAL IN DEVELOPING PRIMARY MIXED-GLIAL CULTURES

In mixed-glial cultures, high concentrations of morphine (1 mu M) have previously been shown to completely inhibit any increase in glial num bers, although DNA synthesis continues in flat, polyhedral astrocytes (type 1 astrocytes). This suggests that high concentrations of morphin e are toxic to glia. Morphine toxicity was assessed in mixed-glial cul tures using calcein-AM and ethidium homodimer dyes as viability marker s to identify live and dead cells, respectively. At 3, 5, and 7 days i n vitro there was no significant difference in the number of dead cell s between untreated and opiate-treated groups. Comparable numbers of e thidium homodimer-labeled cells were present in all groups. The greate st amount of cell death (16-19%) occurred at 3 days in vitro, while fe wer cells (8-12%) were dying at 7 days in vitro. To further characteri ze the dying glia, glial fibrillary acidic protein (GFAP) and A2B5 imm unocytochemistry were combined with viability markers: Only GFAP immun oreactive process-bearing cells and A2B5 immunoreactive cells (process -bearing cells and possibly some neurons) were dying in culture, where as the death of flat, polyhedral GFAP-positive cells was not observed. Cell survival was not affected by morphine, but may be affected by cu lture conditions. Thus, morphine-induced reductions in glial numbers d id not result from an increased rate of cell death. Collectively, the present and previous findings suggest that morphine inhibits the produ ction of flat, polyhedral astrocytes solely by decreasing their rate o f proliferation.