CCh causes a gradual increase in mean synaptic size and a broadening of synaptic size distributions.

<p><b><i>A</i></b>) Example of one experiment. CCh (20 µM) was added to the dish and perfusion media 98 hours after the beginning of the experiment. Panel shows color-coded distributions of fluorescence values of PSD-95:EGFP puncta, for each 0.5 hour time-lapse interval, normalized to the average puncta fluorescence over the 45 hour time window before CCh application. Histograms represent pooled data from all 5 sites in one experiment (same experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040980#pone-0040980-g003" target="_blank">Figure 3</a>). <b><i>B,C</i></b>) Distributions from the experiment shown in <i>A</i>, averaged over 9 hour windows (shown as horizontal lines below <i>A</i>), 4 windows before and 3 windows after CCh application. <b><i>D,E</i></b>) Subtraction of a reference distribution (dashed lines in <i>A–C</i>) from each of the subsequent distributions. <b><i>F</i></b>) Mean fluorescence of PSD-95:EGFP puncta from 6 experiments (36 neurons, 42 sites). Data is shown from 36 hours before to 27 hours after CCh application. Data was normalized separately in each experiment to the average puncta fluorescence of the 36 hour time window before CCh application. To examine the statistical significance of these changes, a paired two tailed t-test was performed for fluorescence values of each time point and each of the 20 pre-CCh time points (dark blue). The grayscale bar above the graph indicates what percentage (legend at the left top) of such tests resulted in statistically significant differences (p<0.05). <b><i>G,H</i></b>) Subtraction of reference distributions as in <i>D</i> and <i>E</i> for the pooled data of these 6 experiments. Asterisks indicate that >99% of Kolmogorov–Smirnov tests performed for each time point in a particular 9 hour time window were significantly different (p<0.01) from each distribution in appropriate reference windows. Data in <i>F-H</i>, mean ± SEM.