The Journal of Neuroscience, February 8, 2006, ():
Integrins Control Dendritic Spine Plasticity in Hippocampal Neurons through NMDA Receptor and Ca2+/Calmodulin-Dependent Protein Kinase II-Mediated Actin Reorganization
J. Neurosci. Shi and Ethell
26: 1813
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Cultured hippocampal neurons showed F-actin reorganization between 7 and 14 DIV concurrent with filopodia transformation into spines.
Confocal images of 7 DIV (A) and 14 DIV (B) mouse hippocampal neurons. Detection of polymerized F-actin with rhodamine-coupled phalloidin. A, 7 DIV hippocampal neurons showed many thin filopodia, which are driven by linear organized F-actin polymers appearing as hair-like extensions along the dendrite (arrowheads), while most F-actin polymers are found within the dendritic shaft. B, At 14 DIV F-actin becomes highly concentrated in dendritic spine heads forming highly branched structures, which appear as intense puncta along the dendrite (arrows). Scale bar: 10 μm in top panels; 5 μm in lower panels.
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Supplemental Figure 2. RGD induced NMDAR activation in 14 DIV hippocampal neurons. A, Western blot analysis of NMDAR (NR2A/B) phosphorylation on tyrosine following RAD or RGD treatment in the absence (left panel) or presence of NMDAR-specific inhibitor MK-801 (right panel). Lysates from treated 14 DIV hippocampal neurons were immunoprecipitated (IP) with anti-NR2A/B antibody. Imunoprecipitates were subsequently immunoblotted (IB) with anti-phosphotyrosine (pY-NR2A/B; upper panels) or anti-NR2A/B (lower panels) antibodies.
B, The level of tyrosine phosphorylation was quantified by densitometry and normalized to total protein level. All values are reported as the mean of three separate experiments ± SD. **, p<0.01 as determined by one way ANOVA.
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Supplemental Figure 3. MK801 blocked RGD-induced and NMDA-mediated dendritic spine remodeling. A-H, Confocal images of GFP-labeled 14 DIV hippocampal neurons from cultures treated with blank (A), RAD (B), RGD (C), NMDA (D), MK801 (E), MK801 plus RAD (F), MK801 plus RGD (G) or MK801 plus NMDA (H). Detection of polymerized F-actin with rhodamine-coupled phalloidin (red) and dendritic morphology with GFP fluorescence (green). NMDAR blockade with its antagonist MK801 (10 μM) prevented both RGD-induced and NMDA-mediated dendritic spine remodeling. Scale bars, 10 μm in top panel; 5 μm in lower panel.
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Supplemental Figure 4. NMDAR blockade with MK801 prevented RGD-induced and NMDA-mediated dendritic spine remodeling.
A, Live images of GFP-labeled dendritic spines in 14 DIV hippocampal neurons before (0 min) and after (60 min) treatment with MK801 plus RGD, MK801 plus NMDA, or NMDA.
B, C, The NMDAR blockade with its antagonist MK801 prevented both RGD-induced and NMDA-mediated the elongation of dendritic protrusions (B) and extension of new filopodia (C). 14 DIV hippocampal neurons were pretreated with MK801 (10 μM). Live images were taken at 3-min intervals for 1 h. Arrows indicate time of RGD or NMDA application, arrowhead indicates time of NMDA removal. Data represent mean protrusion length (B) or average number of protrusions per 10 µm of dendrite (C). Error bars indicate SEM (n = 3 neurons per group). *, p<0.05; **, p<0.01; ***, p<0.001 with one way ANOVA.
