Simultaneous measurements of ratio and AP-evoked SRC1 de-staining rate demonstrate heterogeneity of among synapses supplied by single axons.

<p>(A) Experimental paradigm. A presynaptic cell was filled with the morphological tracer Alexa Fluor 568 using whole-cell patch-clamp. Recycling synaptic vesicles in all boutons in the field of view were labeled with SRC1 using high-frequency field stimulation. After dye washout the ratio in individual boutons was estimated using 30-Hz 60 AP train (see main text and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001396#pbio.1001396.s010" target="_blank">Text S2</a> for details). This was followed by measurements of SRC1 de-staining kinetics () during low-frequency 0.5-Hz stimulation. (B) Fluorescence image from a typical experiment showing SRC1 labeled boutons (red) and axodentritic tree of a single neuron filled with Alexa Fluor 568 (blue). White boxes depict regions of interest containing synaptic boutons analyzed in (C). (C) High resolution images and de-staining profiles in three typical synaptic boutons (depicted by arrows) supplied by the Alexa Fluo 568 loaded neuron. (D and E) Frequency histograms of (D) and (E) from the experiment illustrated in (B and C). Blue histogram, boutons supplied by the Alexa loaded axon; grey histogram, all boutons in the field of view. (F) Comparison of and variability. Blue bars, average CVs for and recorded in synaptic boutons located on single axons; gray bars, average CVs for the same parameters for all boutons recorded in the same experiments. Data are mean ± standard error of the mean (SEM) from 11 independent experiments, **<i>p</i><0.01 and ***<i>p</i><0.001, Wilcoxon signed rank test. (G) Relationship between and . Blue, data points from boutons supplied by the Alexa loaded axon shown in (B); grey data points from all boutons in the field of view. Dotted line shows linear regression for all boutons in the field of view. Pearson's correlation coefficient <i>r</i> and significance levels <i>P</i> (Pearson product correlation test) are indicated. (H) Frequency histogram of calculated for the same set of boutons as in (D, E, and G) using . Scale bars 20 µm (B) and 2 µm (C).