Fig. 4.

cAMP amplifies the spot-like Ca²⁺ transients in the acute dissociated hMFBs. (A) Ca²⁺ current was elicited by the depolarization (Depo.) (0 mV, 10 ms) in control (Top) and with cAMP (0.5 mM) introduced via a patch pipette (Bottom). (B) Fluorescence of Ca²⁺-sensitive dye (OGB6F, 0.2 mM) was loaded via a patch pipette. Images represent the differences between the average intensity during the stimulation (three frames) and the one before stimulation (eight frames), for control (Top) and cAMP conditions (Bottom). Arrowheads indicate the spots where the fluorescence intensity locally increased in response to depolarization. (C) The number of Ca²⁺ spots per bouton against the amplitude of the inward current. These values were not significantly different between control and cAMP conditions (P = 0.81 for the number of spots; 0.67 for the current amplitude; unpaired t test). In C and G–I, control (black) and cAMP (red) data are shown. (D and E) Sequential F/F₀ images at the spot 1 and 1′ in B (D) and of the averaged spots for control and cAMP conditions (E). Five consecutive frames across the depolarization (from 0 to 10 ms, underlined numbers) were depicted. In D–F, the Ca²⁺ spots were centered in a 2 μm × 2 μm cropped square. (F) Schematic view of the measurement of the Ca²⁺ spot. The mean F/F₀s inside the most inner circle (light green; “midmost”) and at the filled areas between the adjacent circles (“annuli”) were measured. The circles have a radius of 0.26, 0.39, 0.52, 0.65, 0.78, and 0.91 μm, from inner to outer, respectively. (G) Sequential F/F₀ changes at the midmost in control and cAMP. Gray dashed lines indicate the onset and the end of depolarization. In G and H, the values are the averages of 23 and 25 spots from five animals for control and cAMP, respectively. (H) F/F₀s are plotted against the distance from the spot center at 5 ms after the onset of depolarization (arrow in G) in control and cAMP. The vertical dotted lines correspond to the radius of the circles in F, and each diamond indicates the mean F/F₀ at the area. (I) Histograms and cumulative graphs of maximum F/F₀ at the midmost for control and cAMP. Values over 3.0 were pooled on the rightest (asterisk). Statistical significance was evaluated by two-sample Kolmogorov–Smirnov test (P = 2.7 × 10⁻⁶). (J) Maximum F/F₀ at the midmost were averaged per bouton for control and cAMP. (K) Maximum F/F₀ in the whole bouton for control and cAMP.