越後街道探索 : その4・諏訪峠 編

departmental bulletin pape

CCh does not induce synaptic growth in the presence of TTX.

<p>Two experiments in which TTX (1 µM) was added to the MEA dishes and the perfusion media 1–2 hours after the experiment was started. <b><i>A</i></b>) Spontaneous activity in each network. <b><i>B</i></b>) Synaptic PSD95:EGFP fluorescence distributions. Whereas TTX applications were followed by a significant broadening of these distributions, CCh had no further effect.</p

Second applications of freshly prepared CCh solutions do not affect activity characteristics or synaptic growth.

<p>Two experiments (out of three in total) in which a second bolus of freshly prepared CCh solution was added to the MEA dishes 143 h (left, same experiment as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040980#pone-0040980-g001" target="_blank">Figure 1</a>) and 137 hours (right) after the experiment was started (45 and 89 hours after first CCh application, respectively). <b><i>A</i></b>) Spontaneous activity rates. <b><i>B</i></b>) Burstiness index. <b><i>C</i></b>) Mean synaptic size (left - 5 sites; right - 8 sites).</p

CCh-induced synaptic growth is not governed by multiplicative scaling or major changes in synaptic remodeling dynamics.

<p><b><i>A</i></b>) Example of fluorescence values for 3 PSD-95:EGFP puncta over a 9 hour time window (thin lines) and after smoothing with a 5-point (2 hour) low pass filter (thick lines). The calculation of <i>ΔF</i> and <i>F₀</i> in subsequent figures is illustrated for the topmost trace. <i>ΔF</i> was calculated by subtracting the fluorescence at the beginning of each time window (F₀) from the fluorescence at its end. As all such measurements were obtained from data smoothed with a 5 point filter, <i>F₀</i> and <i>ΔF</i> were measured from the third data points from the beginning and the end of each 9 hour (18 data point) window. <b><i>B</i></b>) <i>ΔF</i> vs. <i>F₀</i> values for all tracked PSD-95:EGFP puncta in one site during a time window preceding CCh application. Note the significant changes in puncta fluorescence values (even after smoothing as showed in A) and a general trend which can be approximated by a linear regression fit with a negative slope that crosses that abscissa quite far from the origin. <b><i>C,D</i></b>) Averaged <i>ΔF</i> over <i>F₀</i> plots for pooled data (1087 puncta from 10 neurons from 5 separate experiments) before and after CCh application. Individual puncta of 2 neurons per experiment were followed for 54 hours (27 hours before and 27 hours after CCh application). To correct for cell to cell variability in PSD-95:EGFP expression levels, the fluorescence values of each neuron’s synapses were normalized to the average synaptic fluorescence of that neuron during the 27 hour period before CCh application (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040980#s4" target="_blank">Materials and Methods</a> for further details). Thus, normalized <i>ΔF</i> and <i>F₀</i> values are expressed as fractions of mean control fluorescence. As shown in <i>C</i> (inset), distributions of normalized synaptic fluorescence values in almost all neurons were very similar, indicating that this normalization procedure is appropriate. Data was then combined, binned (bin size  = 0.2 mean fluorescence units) and plotted, including only bins that contained at least 45 synapses (∼5% of the data). <b><i>E</i></b>) Distribution of <i>ΔF</i> values for 9 hour windows before and after CCh applications. <b><i>F</i></b>) Changes in the slopes of <i>ΔF</i> vs. <i>F₀</i> regression fits between consecutive time windows before and after CCh application plotted for each cell separately (control → control: −27 to −18 vs. −18 to −9; control → CCh: −9 to 0 vs. 0 to 9). <b><i>G</i></b>) Illustration of the two components presumed to give rise to the overall trends observed in <i>ΔF</i> vs. <i>F₀</i> plots before (left) and after (right) CCh application. Data in <i>C,D</i>, mean ± SEM.</p

Gradual recovery of synchronous bursting activity but not mean synaptic size in the continual presence of CCh.

<p>Pooled data from 4 experiments from 27 hours before to 63 hours after CCh application. <b><i>A</i></b>) Firing rates recorded from all 59 electrodes, normalized to average firing rates during the 27 hour time window before CCh application. <b><i>B</i></b>) Burstiness index. <b><i>C</i></b>) Mean PSD-95:EGFP puncta fluorescence normalized separately in each experiment to average puncta fluorescence during the 27 hour time window preceding CCh application. Grayscale bars indicate statistical significance as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040980#pone-0040980-g002" target="_blank">Figure 2F</a>. Data shown as mean ± SEM.</p

Rapid recovery of synchronous bursting and reversal of synaptic growth is observed after abrupt blocking of cholinergic receptors.

<p>9 hours after the application of CCh, Atropine (1 µM), and Mecamylamine (1 µM) were added to the MEA dishes and the perfusion media. Data pooled from 3 experiments. <b><i>A</i></b>) Burstiness index. <b><i>B</i></b>) PSD-95:EGFP puncta fluorescence distribution (27 sites, 26 neurons) and <b><i>C</i></b>) Mean PSD-95:EGFP puncta fluorescence. <b><i>D</i></b>) Normalized, pooled and binned <i>ΔF</i> vs. <i>F₀</i> plots for individually tracked synapses (674 puncta from 6 neurons from 3 separate experiments) before CCh (dashed line), after CCh application (blue line) and antagonist application (red and green lines). Note that both CCh and the antagonists mainly affected the intercepts of these plots, whereas the slopes were less affected. <b><i>E</i></b>) Changes in the slopes of <i>ΔF</i> vs. <i>F₀</i> linear regression fits between consecutive time windows before CCh, after CCh application and after antagonist application plotted for each cell separately. Note the lack of consistent trends. Data in <i>A,C,D</i>, mean ± SEM.</p

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.</p

Synchronous bursting activity and measures of synaptic remodeling are not strongly correlated.

<p>Data from 8 different experiments (16 tracked sites) in 9 hour windows before (blue) and after (red) CCh application. Lines represent linear regression fits made separately for each group. The four panels show correlations between the burstiness index and <b><i>A</i></b>) changes in mean synaptic PSD95:EGFP fluorescence (each point represents mean change in fluorescence of all synapses of all neurons in a certain experiment), <b><i>B</i></b>) slopes of the <i>ΔF</i> vs. <i>F₀</i> plots (each point represents data from tracked synapses from one site), and <b><i>C</i></b>) the intercepts in the same plots.</p

CCh suppresses synchronous bursting in <i>ex-vivo</i> networks of cortical neurons.

<p>Example of one experiment (same experiment as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040980#pone-0040980-g002" target="_blank">Figure 2A–E</a>). <b><i>A</i></b>) Spontaneous activity recorded from a network of cortical neurons growing on an MEA dish. A 90 hour time window is shown here, starting 53 hours after mounting of the preparation on the combined MEA recording/imaging system. Activity is expressed as action potentials (measured from all 59 electrodes) per second, averaged over 1 min bins. 98 hours after the beginning of the experiment CCh, (20 µM) was added to the dish and the perfusion media. <b><i>B</i></b>) Examples of one-minute long raster plots of activity recorded from 59 electrodes at 80, 99, 118, 142 hours. Note the near elimination of synchronous bursts immediately after CCh application (99 h) Scale bar  = 10 sec. <b><i>C,D</i></b>) Histograms showing the distribution of <i>>/<i><10</i> <i>msec bin</i>>. CCh reduced the fractions of bins characteristic of synchronous bursting activity, that is, bins in which most electrodes were active and bins in which no electrodes were active. The histograms are shown in logarithmic (<i>C</i>) and linear scales (<i>D</i>). <b><i>E</i></b>) Changes in the Burstiness Index <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040980#pone.0040980-Wagenaar1" target="_blank">[39]</a> caused by CCh.</i></p

CCh suppresses synchronous bursting while preserving overall activity levels.

<p>Pooled data from 6 experiments, 27 hours before and 27 hours after CCh application. <b><i>A</i></b>) Firing rates recorded from all 59 electrodes, normalized to average firing rates during the 27 hour time window before CCh application). <b><i>B</i></b>) Burstiness index. Grayscale bars indicate statistical significance as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040980#pone-0040980-g002" target="_blank">Figure 2F</a>. All data points - mean ± SEM.</p