Fig. 1.

Bidirectional regulation of cytoplasmic free calcium concentration near the neuronal plasma membrane by β-adrenergic receptors. (A) Dissociated rat hippocampal neurons (DIV12–21) were loaded with Fluo-4 and imaged sequentially every 100 ms by TIRFM. Fluorescence intensity is displayed in pseudocolor (scale palette on the left) before (5 s) and after (15 s) bath application of 10 μM isoproterenol (added at t = 10 s relative to the time stamp in the lower right corner). (B) Time course of the calcium indicator fluorescence intensity measured in the cell body of 10 neurons and plotted versus time. The black line indicates mean intensity, and gray shading represents SEM. The red line represents the presence of isoproterenol during the indicated times. (C) Average fluorescence intensity measured before and after the addition of 10 μM propranolol. The blue line indicates the presence of propranolol (n = 7). (D) The presence of 10 μM alprenolol, a specific β-blocker, prevented the effect of isoproterenol (red line) on calcium indicator fluorescence (n = 6). (E) Analysis across independent experiments of the effect of norepinephrine (NE; 10 μM) and isoproterenol (Isop; 10 μM) on calcium indicator fluorescence measured at 5 s after bath application. Preincubation of neurons (1 min) with 10 μM alprenolol blocked the norepinephrine-, isoproterenol-, and propranolol-induced changes in calcium indicator fluorescence. (F) Preincubation with 50 μM CGP-20712, a concentration that effectively blocks β₁- and β₂-adrenergic receptors, also prevented the calcium-signaling effects of norepinephrine, isoproterenol, and propranolol. Preincubation with 50 nM CGP-20712 (a concentration that selectively blocks β₁-adrenergic receptors) did not prevent isoproterenol- or propranolol-induced changes in calcium indicator fluorescence. (G) Dose–response curve of the isoproterenol-induced decrease in calcium indicator fluorescence measured at 5 s after bath application, normalized to the maximum response. (H) Dose–response curve of the propranolol-induced increase in calcium indicator fluorescence normalized to the maximum response.