Fig. 8: Inhibition pf PLC prevents the return of PIP2 levels to steady-state levels in phase 3 of LTD induction.

a Scheme visualizing the degradation of PIP₂ yielding DAG + IP₃ and the inhibition by the PLC inhibitor U-73122. b–g TEM images of dendritic spines of anti-PIP₂ immunolabeled, freeze-fractured spines of DIV14-16 hippocampal neurons, which were merely pretreated with vehicle control (0.2% DMSO) (−U-73122, b–d) or with 10 µM U-73122 (e–g) prior to either inducing LTD by 3 min NMDA and 7 min recovery time (3 + 7 min) and by 3 min NMDA and 27 min recovery time (3 + 27 min), respectively, or leaving the cells at steady state (0 min). Bars, 200 nm. h Quantitative analyses of the anti-PIP₂ labeling density of + U-72122 and −U-73122 neurons at steady state normalized to the respective untreated control. (i, j) Quantitative analyses of PIP₂ dynamics demonstrating that PLC inhibition has no effect on PIP₂ levels during the second phase (3 + 7 min) of LTD induction but completely blocks the restauration of steady-state PIP₂ levels during the third phase (3 + 27 min) of PIP₂ dynamics during LTD induction. Data, mean ± SEM. −U-73122, 0 min, n = 28; 3 + 7 min, n = 27; 3 + 27 min, n = 32 ROIs each (total spines; spine heads) and +U-73122, 0 min, n = 27; 3 + 7 min, n = 25; 3 + 27 min, n = 26 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 ±U-73122 conditions (i, j). *P < 0.05; **P < 0.01. Additional Mann–Whitney tests for comparisons of +U-73122 and −U-73122 data at 3 + 7 min and 3 + 27 min to 0 min control data (i, j). ^#P < 0.05; ^##P < 0.01; ^###P < 0.001. P-values are reported directly in the figure. For numerical source data, see Supplementary Data 1.