Figure 6.

GluR1-C-tail peptide becomes localized to PSDs after LTP. A, Postembedding immunogold electron microscopy performed against GFP-GluR1 C-tail in slices with or without chemLTP induction. All spines containing a gold particle were identified, and the minimal distance between the gold particle and PSD was measured (see Materials and Methods). A1, Cumulative distribution of all particles within 100 nm of the PSD shown (p < 0.01) (without chemLTP, n = 2 slices, 86 particles; with chemLTP, n = 2 slices, 101 particles). A2, Example images of spines from slices with and without chemLTP induction. Gold particles are marked by red arrowheads. Scale bar, 250 nm. B, Photoconversion of tDimer-GluR1-C-tail used to measure the peptides stability within spines both before and after chemLTP induction. B1, Experiment timeline. Two identical image series are performed, one before and one after chemLTP induction (blue bar). Images acquired at times marked by black triangles. MECC, the photoconversion, performed at times marked by red lines. MECC permanently converts tDimer from a red to a green fluorophore. B2, Spine green/red ratio plotted relative to MECC. Values are normalized to mean baseline values (*p < 0.01, for tDimer-GluR1-C-tail after chemLTP only). Photoconversion is performed on a 25 μm stretch of dendrite so the decay of the green/red ratio during the first 10 min represents mixing of that dendritic region with the rest of the cell and is not an accurate measure of spine to dendrite diffusion. tD-GluR1-C-tail before chemLTP, n = 42 spines, 3 cells; tD-GluR1-C-tail after chemLTP, n = 32 spines, 3 cells; tD only before chemLTP, n = 42 spines, 3 cells; tD only after chemLTP, n = 45 spines, 3 cells. B3, Example images of a spine before and after photoconversion both before and after chemLTP. Images are a pixelwise green/red ratio of two to three collapsed stacks where green represents high G/R, and red represents low G/R. Scale bar, 0.5 μm.