Fig. 7: PIP5K inhibition prevents the PIP2 increase during both the first and the second phase of LTD induction.
a Scheme visualizing suggested routes towards PIP2 during LTD and the inhibition by the PIP5K inhibitor UNC3230. b–g TEM images of dendritic spines of anti-PIP2 immunolabeled, freeze-fractured spines of DIV14-16 hippocampal neurons, which were pretreated with vehicle control (0.002% DMSO) (−UNC3230, b–d) or with 500 nM UNC3230 (e–g) prior to either inducing LTD by 2 min NMDA (50 µM) or with 3 min NMDA and 7 min recovery time (3 + 7 min), respectively, or cryopreserving the cells at steady state (0 min). Bars, 200 nm. h Quantitative analyses of the anti-PIP2 labeling density of +UNC3230 and −UNC3230 neurons at steady state normalized to the respective untreated control. i, j Quantitative analyses of PIP2 dynamics revealing a very strong negative impact of PIP5K inhibition on both the first (2 min) and the second (3 + 7 min) phase of elevated PIP2 signals during LTD induction. Data, mean ± SEM. −UNC3230, 0 min, n = 62; 2 min, n = 46; 10 (3 + 7) min, n = 35 ROIs each (total spines; spine heads) and + UNC3230, 0 min, n = 50; 2 min, n = 40; 10 (3 + 7) min, n = 34 ROIs each (total spines; spine heads) of primary neurons from 3 independent assays. Mann–Whitney (h; n.s.); Two-way ANOVA/Bonferroni’s Multiple Comparison between ±UNC3230 conditions (i, j) *P < 0.05; **P < 0.01. Additional Kruskal–Wallis/Dunn’s Multiple Comparison for comparison of +UNC3230 and −UNC3230 data at 2 min and 3 + 7 min to 0 min control data (i, j)#.P < 0.05; ##P < 0.01. P-values are reported directly in the figure. For numerical source data, see Supplementary Data 1.
