Figure 6

Blockade of adenosine A _2A receptors prevents interleukin (IL)-1β-induced exacerbation of glutamate-mediated calcium deregulation in hippocampal neurons. Rat hippocampal neurons were loaded with the calcium probe Fura-2-AM, and the fluorescence emitted at 510 nm upon alternate excitation at 340 and 380 nm (F₃₄₀ and F₃₈₀, respectively) was monitored in cell bodies every second for 35 minutes. Basal fluorescence ratio was measured for the first 2 minutes, then drugs, prepared in normal Krebs solution, were added to the superfusion using a fast-pressurized system, as indicated by the arrows in (A). When used, 100 ng/ml IL-1β was added for 5 minutes before addition of 100 μmol/L glutamate (Glu). Cells were left in the presence of the stimuli for the next 15 minutes, and then washed through superfusion of normal Krebs. The A_2A receptor selective antagonist, SCH58261 (50 nmol/l), was incubated for 20 minutes before the beginning of the experiment and continued throughout. The results are presented as the ratio (F₃₄₀/F₃₈₀) of recordings of healthy neurons (evaluated by their response to 50 mmol/l KCl, after washing away the stimuli). IL-1β increased both (B) glutamate-induced calcium entry (peak value of the difference between the basal ratio and the stimuli-induced ratio) and (C) glutamate-induced calcium deregulation, calculated as the slope from peak values until the removal of the stimuli. (B) SCH58261 attenuated the exacerbation by IL-1β of glutamate-induced calcium and (C) prevented the calcium deregulation induced by the combination of IL-1β plus glutamate. However, SCH58261 seemed to aggravate the effect of glutamate alone. (D) Representative fluorescence images of the neuronal cell bodies (first row) in basal conditions, (middle row) in the presence of stimuli, and (bottom row) immediately before washing away the stimuli. The color scale of blue, green, yellow, red, and white corresponds to increasing calcium concentrations. (E) Ability of the p38 inhibitor (SB 203580, 10 μmol/L) to attenuate the exacerbation by IL-1β of glutamate-induced calcium entry, and (F) ability of both inhibitors to either attenuate (JNK inhibitor) or prevent (p38 inhibitor) calcium deregulation, whereas neither of the inhibitors by itself affected the dynamics of intracellular calcium. Data are mean ± SEM of four to six different cell cultures (480 to 1200 cells analyzed per condition for each n), *P < 0.05, **P < 0.01.