AbstractSynaptic plasticity involves proper establishment and rearrangement of structural and functional microdomains. Yet, visualization of the underlying lipid cues proved challenging. Applying a combination of rapid cryofixation, membrane freeze-fracturing, immunogold labeling and electron microscopy, we visualize and quantitatively determine the changes and the distribution of phosphatidylinositol-4,5-bisphosphate (PIP₂) in the plasma membrane of dendritic spines and subareas thereof at ultra-high resolution. These efforts unravel distinct phases of PIP₂ signals during induction of long-term depression (LTD). During the first minutes PIP₂ rapidly increases in a PIP5K-dependent manner forming nanoclusters. PTEN contributes to a second phase of PIP₂ accumulation. The transiently increased PIP₂ signals are restricted to upper and middle spine heads. Finally, PLC-dependent PIP₂ degradation provides timely termination of PIP₂ cues during LTD induction. Together, this work unravels the spatial and temporal cues set by PIP₂ during different phases after LTD induction and dissects the molecular mechanisms underlying the observed PIP₂ dynamics.