Reproducible Ca2+ responses at individual presynaptic varicosities evoked by brief tetanic stimulation. A. The recorded MSN is shown as a projection image created from a confocal z series of Alexa594 images. The area outlined (black rectangle) corresponds to the area imaged in panel B. B. OG1 fluorescence image showing the stretch of axon contained one hot spot (arrowhead). The spline used is shown in red, superimposed on the OG1 image. C. Spline scan images. Top image shows Alexa594, and bottom OG1. The timing of repetitive depolarizing pulses to evoke 10 action potentials is indicated on the time line (bottom). As compared to the Alexa594 trace, the OG1 trace shows a stimulation-dependent increase in fluorescence intensity, which is shown in the line graphs in the next panel. D. Ca2+ imaging was repeated under control conditions (without drug application) at 10 min intervals, as diagramed on the time line. Raw traces are shown in blue, and smoothed traces (moving average of ten, see Methods) are superimposed in black. The total duration of the experiment was 40 min. The magnitude of the subsequent Ca2+ responses (measured as area under the curve, a.u.c.) expressed as a percent of the initial response is indicated below each trace. The Ca2+ responses varied less than 5%. E. To test reproducibility, Ca2+ responses were evoked twice under control conditions (no drugs) and the a.u.c. of the second response was expressed as the percent of the first response (n = 9). The second response did not differ significantly from the first (see Results).