The Journal of Neuroscience, March 29, 2006, ():
Stabilization of Axon Branch Dynamics by Synaptic Maturation
J. Neurosci. Ruthazer et al.
26: 3594
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1: Algorithm for automatic punctum identification. A) unfiltered projection of two-photon z-series stack of SYP-CFP (green) and EYFP (red). B) The image is median filtered using a 3-by-3 pixel filter and any zero pixels are set to a value of 1 to reduce noise and permit relative thresholding (20% increase in intensity). C) Individual axon branch tips are drawn in 3-D to provide a template for analysis. D) The branch tips are straightened by dividing the branch into small line segments and arraying them linearly. This permits relative intensity changes to be measured along the length of the axon branch tips. Puncta satisfying the criteria in E (local intensity increase and then decrease within 2µm of each other) are identified and measured by integrating the total fluorescence within a region 7 pixels wide and 6 pixels tall around the identified punctum center. Color bars indicate punctum intensities in relative intensity units. These values are normalized to permit comparison of animals with different labeling intensities by linearly scaling the 80%ile brightest punctum to a value of 80 intensity units. E) Flowchart of punctum identification algorithm.
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Supplementary Figure 2: Stability of synaptic puncta correlates with their intensity SYP puncta that will be lost over eight hours of imaging start out less intense than stable puncta. *** p<0.001, two-tailed t-test.
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Supplementary Figure 3: Punctum intensities from retracting axonal branch tips in unstimulated tadpoles. Punctum intensities are lower in the eliminated part of the branch tip than at the point to which the branches retracted following 4 hr in darkness without visual stimulation N =16 axons, * p<0.05, Kruskal-Wallis with Dunn Post-test)
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Supplementary Movie: Time-lapse movie of axons from figure 2. These axons are expressing SYP-CFP (green) and EYFP (red). Two photon z-series stacks were collected at 1µm intervals through the entire arbor every 10 minutes for a total of two hours.
