Figure 7.
Temporal regulation of CREB shut-off. A, Cultures were costimulated with NMDA (50 μm) and synaptic activity [bicuculline (bic) plus 4-AP with nimodipine and CNQX] from t = 0-10 min. After stimulation, the medium was changed to a TTX-containing solution, and the pCREB expression was determined at the time points outlined. Mock stimulation is shown for the t = 0 time point. Under this 10 min stimulus condition, pCREB expression persisted for 90 min. B, Cultures initially were stimulated (10 min) with synaptic activity (bicuculline plus 4-AP with nimodipine and CNQX from -40 to -30 min) and then placed in a TTX/HEPES buffer (30 min; from -30 to 0 min). After this treatment, NMDA (50 μm) was administered for 10 min (from 0 to 10 min), and the cells were then returned to a TTX/HEPES buffer. pCREB expression was determined at the time points outlined. pCREB levels for both synaptic stimulation (bicuculline plus 4-AP in the presence of nimodipine and CNQX) and mock stimulation [control (Con)] are shown for the t = 0 time point (before NMDA administration). Under this sequential stimulus paradigm, pCREB expression returned to basal levels by 25-45 min after NMDA administration. Data are representative of triplicate determinations.