FIGURE 1.

A–H, preconditioning rat cortical neurons with 4-aminopyridine/bicuculline induces tolerance to otherwise lethal OGD. Neurons were subjected to sham wash or to preconditioning by 2.5 mm 4-AP plus 50 μm bicuculline (4-AP/bic) for 48 h, followed by a 24-h recovery period, then subjected to control wash or 65–80 min OGD, followed by assessment of neurotoxicity by measurement of the % PI-uptake 24 h later. Same-field phase contrast and PI fluorescence image pairs were acquired from the following conditions: A and B, sham wash; C and D, 4-P/bic preconditioning, followed by OGD; E and F, OGD; or G and H, 100% neuronal kill achieved by subjecting sister cultures to 100 μm NMDA for 15 min. I, preconditioning increases neuronal activity. Neuronal intracellular Ca²⁺ spiking in Fluo-4-loaded neurons 0.5–2.5 h following application of sham wash, 50 μm bicuculline (bic), 20 μm 4-aminopyridine plus 50 μm bicuculline (4-AP/bic), or to 4AP/bic applied with a Na⁺ channel blocker, tetrodotoxin (TTX, 1 μm). J–P, a wide range of 4-AP/bic concentrations and duration of exposure, as well as the duration of the recovery interval between preconditioning and OGD, result in neuroprotection. Cultures were subjected to sham wash or to preconditioning with 50 μm bicuculline combined with 4-AP (20, 300, or 2500 μm) for 4–48 h, followed by washout and recovery for 0–96 h, subsequently exposed to 65–80 min OGD, followed by measurement of % PI-uptake 24 h later (n = 12–18). Preconditioning resulted in a significant suppression of % PI-uptake following OGD (p < 0.05; calculated using an ANOVA), except for the shortest 4-AP/bic duration investigated (4 h; Fig. 1P) or for the longest interval between preconditioning and OGD investigated (96 h; Fig. 1J).