Fig. 9.

Changes in concentration of extracellular Ca²⁺ that affect CaMKII autophosphorylation produced no detectable change in intracellular concentration in DRG neurons.A, Action potentials induced by brief electrical stimulation (10 Hz) caused a large increase in intracellular calcium as measured with fluo-3 fluorescence in time-lapse confocal microscopy. After intracellular calcium concentration recovered to basal levels, the extracellular calcium concentration was lowered from 1.2 mm Ca²⁺ to 50 nmCa²⁺ using a rapid bath perfusion system (Low Ca²⁺). The intracellular calcium concentration was not altered significantly (a), and remained constant over the next 25 min (b). Electrical stimulation produced no change in intracellular calcium in low calcium conditions (b, inset, 10 Hz), indicating that the change to low extracellular calcium concentration by perfusion of 50 nm Ca²⁺ solution had been effective. After then returning extracellular calcium concentration to 1.2 mm (Ca²⁺stim), intracellular calcium concentration remained constant for the next 40 min (b, inset). Calcium ionophore A23187 applied at the end of the experiment (*) caused a large increase in intracellular calcium, demonstrating the efficacy of the measurement technique. B, Similar results were obtained using the ratiometric calcium indicator indo-1, to correct for decreasing fluorescence caused by photobleaching in prolonged recordings and to obtain a quantitative estimate of changes in intracellular calcium. Brief 10 Hz stimulation or depolarization with 90 mm KCl caused large increases in intracellular Ca²⁺ concentration, but intracellular Ca²⁺ levels remained constant after lowering extracellular Ca²⁺ concentration from 1.2 mm to 50 nm or increasing extracellular Ca²⁺ from 50 nm to 1.2 mm (Ca²⁺stim). Results shown are mean ± SEM;n = 8 neurons in A and 13 neurons inB. Although extracellular Ca²⁺stimulation did not alter intracellular calcium measurably, the changes in extracellular calcium caused large changes in autonomous activity of CaMKII (Fig. 8).