Chronic hypoxia activates apoptosis in dissociated neuronal cultures. (A) Continuous hypoxia induces nuclear pyknosis and caspase-3 cleavage in DIV7 cortical neurons. Neuronal cultures were exposed to hypoxia (0.5% O2) for 18 h and analyzed by fluorescence microscopy for expression of the neuronal marker NeuN, nuclear pyknosis using the DNA dye Hoechst 33342, and the cleavage of caspase-3. (B) Nuclear pyknosis approximates caspase-3 cleavage in vitro. (C) Hypoxia-induced neuron death requires de novo translation. Cultures were pretreated with cycloheximide (CHX 1 μg/ml, 3.5 μM) or a combination of the glutamate receptor antagonists MK-801 (100 μM) and CNQX (10 μM), and exposed hypoxia (0.5% O2, 18 h; filled bars) and analyzed for nuclear pyknosis versus control (21% O2; open bars) sister cultures. Data represent the average±S.D. of results from six non-overlapping fields across replicates (n=4; *=P<0.05).