Fig. 3: PIP2 transiently increases after LTD induction in dendritic spines.
a–h TEM images of anti-PIP2 immunogold-labeled dendritic spines (outlined) of freeze-fractured hippocampal neurons (DIV14-16) at steady state (0 min) and at different times during and after LTD induction by incubation with 50 µM NMDA for 3 min and subsequent placing back into preconditioned medium. Bars, 200 nm. i–k Quantitative determinations of anti-PIP2 immunogold labeling densities at different treatment times during and after LTD induction determined in dendrites (i), in total spines (j) and in the three different spine subdomains (k) expressed as percent of the average labeling density of the total spine data at 0 min (steady state) in each assay. Three apparent phases of PIP2 signaling responses in dendritic spines during and after LTD induction are highlighted by coloring (yellow, phase 1, rapid first increase of PIP2 density; green, second phase of increase of PIP2 signals in the spine (head) plasma membrane until a maximum is reached at 10 min; blue, phase 3, subsequent decreasing PIP2 signals in the spine (head)). For a comparison of absolute data for 0 and 3 + 7 min not normalized to the respective controls with corresponding data obtained from an independent, untrained experimenter see Supplementary Fig. 3. (i–k) Data, mean ± SEM. 0 min, n = 159; 1 min, n = 36; 2 min, n = 53; 3 min, n = 49; 5 (3 + 2) min, n = 50; 10 (3 + 7) min, n = 59; 15 (3 + 12) min, n = 44; 30 (3 + 27) min, n = 35 ROIs each (dendrite; total spine; spine subdomains, base, neck, and head) from 3-16 independent assays with different incubation times. Statistical significance calculations, Kruskal–Wallis/Dunn’s (i–k; **P < 0.01; ****P < 0.0001) and Mann–Whitney tests for 3 vs. 10 min and 10 vs. 30 min (j; ##P < 0.01) (j). For P < 0.0001, exact P-values are not available. Other P-values are reported directly in the figure. For numerical source data, see Supplementary Data 1.
