Co-variation of and volume averaged AP-evoked presynaptic Ca²⁺ fluorescent transient.

<p>(A) Fluorescence image (Alexa channel) of the same neuron as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001396#pbio-1001396-g003" target="_blank">Figure 3B</a> after completion of the SRC1 de-staining experiment and subsequent re-patch-loading with Fluo-4 and Alexa Fluor 568. (B) Experimental paradigm. Ca²⁺ fluorescence responses in each bouton were recorded during a single sweep consisting of five APs delivered at 0.5 Hz followed by a saturating 100-Hz train of APs. The laser was turned on only during 100-ms intervals synchronized with single AP stimulation (brown arrows) and at the very end of the 100-Hz 100 AP train (brown horizontal bar) when Fluo-4 signal was already saturated (e.g., <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001396#pbio-1001396-g001" target="_blank">Figure 1G</a>). (C) Line-scan recordings of Ca²⁺ dynamics in three typical boutons (prior to these recordings vesicular release in these boutons was measured as illustrated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001396#pbio-1001396-g003" target="_blank">Figure 3C</a>). The morphology of each bouton (Alexa channel) and the position of line scans are shown on the left. The aspect ratio in line-scan images has been adjusted to optimize figure layout. (D) Amplitude of AP-evoked presynaptic Ca²⁺ influx does not change during 0.5-Hz stimulation. In each recorded bouton Ca²⁺ fluorescence response at every AP of the 0.5-Hz train (Δ<i>F</i>(AP)) was normalized to the average amplitude calculated for all five APs (<Δ<i>F</i>>) in the same train. The bars are the mean ± SEM values of Δ<i>F</i>(<i>AP</i>)/<Δ<i>F</i>> in all recorded boutons (<i>n</i> = 42 from ten independent experiments). Δ<i>F</i>(<i>AP</i>)/<Δ<i>F</i>> did not vary systematically with the AP number (<i>p</i> = 0.2, one-way ANOVA). (E–G) AP-evoked SRC1 de-staining rate (D), the average release probability of individual RRP vesicles (E), and the ratio (F), plotted against the amplitude of AP-evoked presynaptic Ca²⁺ fluorescence Δ<i>F/F_m</i>. All parameters are normalized to the respective mean value in each experiment (<i>n</i> = 42 boutons from ten independent experiments). Correlation coefficients ρ and significance levels <i>p</i> (Spearman rank correlation test) are indicated. Dashed lines show data fits with a power function. Color-coded data points correspond to boutons 1, 2, and 3 from the experiment analyzed in (C) and also in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001396#pbio-1001396-g003" target="_blank">Figure 3B and 3C</a>. Scale bars 20 µm (A) and 2 µm (C).