The Journal of Neuroscience, October 5, 2005, ():
Stereotyped Axon Pruning via Plexin Signaling Is Associated with Synaptic Complex Elimination in the Hippocampus
J. Neurosci. Liu et al.
25: 9124
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1: Serial 3D reconstructions of mossy fiber synaptic complexes in the IPB before and during the course of IPB pruning. (A) Electron micrograph of a P15 WT mossy fiber bouton (green) taken before pruning of the IPB that establishes many asymmetric synaptic contacts (yellow) with a dendritic shaft and its spines (pink). The micrograph was taken from one of 15 thin sections (70 nm per section) from the 3D serial reconstruction in (B) (gray square). (B) 3D serial reconstruction of the same P15 WT mossy fiber bouton in association with a CA3 dendrite shaft and a bifurcated spine branching off of the dendritic shaft. (C, E, G) Electron micrographs of much smaller boutons taken during pruning of the IPB from P20 and P25 WT mice from the 3D serial reconstructions in (D) (17 thin sections, 70 nm per section) and (F) (12 thin sections, 70 nm per section), and (H) (14 thin sections, 70 nm per section), respectively. (D, F, H) 3D serial reconstructions of the same smaller boutons at P20 and P25 WT. The boutons are much smaller than at P15 and are shown here each making only one asymmetric synaptic contact with a dendritic spine. Scale bars: 0.5 mm
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Supplementary Figure 2: Characterization of mossy fiber synaptic complexes of the MB with synaptic markers. (A) P15 WT, an immunoelectron micrograph shows VGLUT2 immunoreactivity in association with synaptic vesicles in a mossy terminal-like bouton (t) from the MB forming multiple synaptic contacts with spines (sp) and a dendritic shaft (d). (B) P15 WT, an immunoelectron micrograph shows NMDAR1 immunoreactivity in association specifically with postsynaptic densities (arrowhead) localized in a dendritic shaft (d) which is contacted by a mossy terminal-like bouton (t) from the MB. (C) P45 WT, a mossy terminal-like bouton (t) from the MB filled with dense VGLUT1 immunolabeled vesicles makes multiple synaptic contacts with spines (sp) and dendrites (d). (D) P45 WT, a large mossy terminal-like bouton (t) from the MB makes synaptic contacts with NMDAR1-immunolabeled dendrites (d) and spines (sp); note that PSDs (arrowhead) are in association specifically with NMDAR1 immunoreactivity. Scale bars: 0.2 mm.
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Supplementary Figure 3: Localization of Timm’s staining and CB immunolabeling in alternate serial sections of P20 and P25 WT mice. (A, B) P20 WT serial sections, an intermediate stage of pruning where CB labeling of mossy fibers persists while the level of Timm’s staining for mossy fiber boutons disappears. (C, D) P25 WT serial sections, a more completed stage of pruning shows that the level of CB and Timm’s staining in the IPB is approximately equal, as the mossy fibers of the IPB have pruned back most of their transient projections to join the MB projections. White arrowheads indicate the level of Timm’s or CB labeling in the IPB. (E, F) P25 PLXA3 knockout serial sections, demonstrating that the level of CB and Timm’s staining in the IPB is approximately equal for a case where pruning does not occur. Scale bar for (A-F): 200 mm
(G) Bar graph showing the average normalized ratio (mean ± S.E.) of CB to Timm’s staining for mossy fiber development in the IPB. Since pruning is fast and can occur at any time between P20-P30, samples taken from P20-P25 mice have been placed into three groups based on their relative degrees of pruning; this is with referenceto what has been quantified for the IPB of WT mice in our previous paper (Bagri et al., 2003): ‘before pruning’, ‘during pruning’, ‘after pruning’. In cases where the IPB was not pruned or mostly pruned away—P15 WT (n = 3 mice), P20-P25 WT ‘before pruning’ (n = 10 mice), P20-P25 WT ‘after pruning’ (n = 5 mice), and P25 PLXA3 knockout samples (n = 4 mice), respectively—the level of CB to Timm’s staining is approximately equal. During pruning for P20-P25 WT ‘during pruning’ samples (n = 5 mice) (*), the level of CB labeling is almost 3-fold longer than Timm’s staining in the IPB. Significant differences between means were observed only between P20-P25 WT ‘during pruning’ versus P15 WT, P20-P25 WT ‘before pruning’, P20-P25 WT ‘after pruning’, and P25 PLXA3 knockout samples (p < 0.001; ANOVA, Newman-Keuls test).
